Embryonic-like stem cells derived from post-partum mammalian placenta, and uses and methods of treatment using said cells

ABSTRACT

The present invention provides compositions and methods of using embryonic-like stem cells that originate from a post-partum placenta with conventional cord blood compositions or other stem or progenitor cells. The embryonic-like stem cells can be used alone or in a mixture with other stem cell populations. In accordance with the present invention, the embryonic-like stem cells may be mixed with other stem cell populations, including but not limited to, umbilical cord blood, fetal and neonatal hematopoietic stem cells and progenitor cells, human stem cells and progenitor cells derived from bone marrow. The embryonic-like stem cells and the mixed populations of embryonic-like stem cells and stem cells have a multitude of uses and applications, including but not limited to, therapeutic uses for transplantation and treatment and prevention of disease, and diagnostic and research uses.

This application is a continuation-in-part of application Ser. No.10/076,180, filed Feb. 13, 2002 now abandoned, which is herebyincorporated by reference herein. The present application also claimspriority to U.S. provisional application No. 60/437,292, filed Dec. 31,2002, which is hereby incorporated by reference herein.

1. INTRODUCTION

The present invention relates to the use of embryonic-like stem cellsthat originate from a post-partum placenta with conventional cord bloodcompositions or other stem or progenitor cells. The embryonic-like stemcells can be used alone or in a mixture with other stem cellpopulations. In accordance with the present invention, theembryonic-like stem cells may be mixed with other stem cell populations,including but not limited to, umbilical cord blood, fetal and neonatalhematopoietic stem cells and progenitor cells, human stem cells andprogenitor cells derived from bone marrow. The embryonic-like stem cellsand the mixed populations of embryonic-like stem cells and stem cellshave a multitude of uses and applications, including but not limited to,therapeutic uses for transplantation, diagnostic and research uses. Theembryonic-like stem cells and the mixed populations are also useful inthe treatment of diseases or disorders, including vascular disease,neurological diseases or disorders, autoimmune diseases or disorders,diseases or disorders involving inflammation, and cancer or thedisorders associated therewith. In particular, the embryonic-like stemcells or mixtures including them are administered in high doses andwithout HLA typing.

2. BACKGROUND OF THE INVENTION

There is considerable interest in the identification, isolation andgeneration of human stem cells. Human stem cells are totipotential orpluripotential precursor cells capable of generating a variety of maturehuman cell lineages. This ability serves as the basis for the cellulardifferentiation and specialization necessary for organ and tissuedevelopment.

Recent success at transplanting such stem cells have provided newclinical tools to reconstitute and/or supplement bone marrow aftermyeloablation due to disease, exposure to toxic chemical and/orradiation. Further evidence exists that demonstrates that stem cells canbe employed to repopulate many, if not all, tissues and restorephysiologic and anatomic functionality. The application of stem cells intissue engineering, gene therapy delivery and cell therapeutics is alsoadvancing rapidly.

Many different types of mammalian stem cells have been characterized.For example, embryonic stem cells, embryonic germ cells, adult stemcells or other committed stem cells or progenitor cells are known.Certain stem cells have not only been isolated and characterized buthave also been cultured under conditions to allow differentiation to alimited extent. A basic problem remains, however, in that obtainingsufficient quantities and populations of human stem cells which arecapable of differentiating into all cell types is near impossible. Stemcells are in critically short supply. These are important for thetreatment of a wide variety of disorders, including malignancies, inbornerrors of metabolism, hemoglobinopathies, and immunodeficiencies. Itwould be highly advantageous to have a source of more embryonic stemcells.

Obtaining sufficient numbers of human stem cells has been problematicfor several reasons. First, isolation of normally occurring populationsof stem cells in adult tissues has been technically difficult and costlydue, in part, to very limited quantity found in blood or tissue.Secondly, procurement of these cells from embryos or fetal tissue,including abortuses, has raised religious and ethical concerns. Thewidely held belief that the human embryo and fetus constituteindependent life has prompted governmental restrictions on the use ofsuch sources for all purposes, including medical research. Alternativesources that do not require the use of cells procured from embryonic orfetal tissue are therefore essential for further progress in the use ofstem cells clinically. There are, however, few viable alternativesources of stem cells, particularly human stem cells, and thus supply islimited. Furthermore, harvesting of stem cells from alternative sourcesin adequate amounts for therapeutic and research purposes is generallylaborious, involving, e.g., harvesting of cells or tissues from a donorsubject or patient, culturing and/or propagation of cells in vitro,dissection, etc.

For example, Caplan et al. (U.S. Pat. No. 5,486,359 entitled “Humanmesenchymal stem cells,” issued Jan. 23, 1996), discloses humanmesenchymal stem cell (HMSC) compositions derived from the bone marrowthat serve as the progenitors for mesenchymal cell lineages. Caplan etal. discloses that hMSCs are identified by specific cell surface markersthat are identified with monoclonal antibodies. Homogeneous hMSCcompositions are obtained by positive selection of adherent marrow orperiosteal cells that are free of markers associated with eitherhematopoietic cell or differentiated mesenchymal cells. These isolatedmesenchymal cell populations display epitopic characteristics associatedwith mesenchymal stem cells, have the ability to regenerate in culturewithout differentiating, and have the ability to differentiate intospecific mesenchymal lineages when either induced in vitro or placed invivo at the site of damaged tissue. The drawback of such methods,however, is that they require harvesting of marrow or periosteal cellsfrom a donor, from which the MSCs must be subsequently isolated.

Hu et al. (WO 00/73421 entitled “Methods of isolation, cryopreservation,and therapeutic use of human amniotic epithelial cells,” published Dec.7, 2000) discloses human amniotic epithelial cells derived from placentaat delivery that are isolated, cultured, cryopreserved for future use,or induced to differentiate. According to Hu et al. a placenta isharvested immediately after delivery and the amniotic membrane separatedfrom the chorion, e.g., by dissection. Amniotic epithelial cells areisolated from the amniotic membrane according to standard cell isolationtechniques. The disclosed cells can be cultured in various media,expanded in culture, cryopreserved, or induced to differentiate. Hu etal. discloses that amniotic epithelial cells are multipotential (andpossibly pluripotential), and can differentiate into epithelial tissuessuch as corneal surface epithelium or vaginal epithelium. The drawbackof such methods, however, is that they are labor-intensive and the yieldof stem cells is very low. For example, to obtain sufficient numbers ofstem cells for typical therapeutic or research purposes, amnioticepithelial cells must be first isolated from the amnion by dissectionand cell separation techniques, then cultured and expanded in vitro.

Umbilical cord blood (“cord blood”) is a known alternative source ofhematopoietic progenitor stem cells. Stem cells from cord blood areroutinely cryopreserved for use in hematopoietic reconstitution, awidely used therapeutic procedure used in bone marrow and other relatedtransplantations (see e.g., Boyse et al., U.S. Pat. No. 5,004,681,“Preservation of Fetal and Neonatal Hematopoietin Stem and ProgenitorCells of the Blood”, Boyse et al., U.S. Pat. No. 5,192,553, entitled“Isolation and preservation of fetal and neonatal hematopoietic stem andprogenitor cells of the blood and methods of therapeutic use”, issuedMar. 9, 1993). Conventional techniques for the collection of cord bloodare based on the use of a needle or cannula, which is used with the aidof gravity to drain cord blood from (i.e., exsanguinate) the placenta(Boyse et al., U.S. Pat. No. 5,192,553, issued Mar. 9, 1993; Boyse etal., U.S. Pat. No. 5,004,681, issued Apr. 2, 1991; Anderson, U.S. Pat.No. 5,372,581, entitled Method and apparatus for placental bloodcollection, issued Dec. 13, 1994; Hessel et al., U.S. Pat. No.5,415,665, entitled Umbilical cord clamping, cutting, and bloodcollecting device and method, issued May 16, 1995). The needle orcannula is usually placed in the umbilical vein and the placenta isgently massaged to aid in draining cord blood from the placenta.Thereafter, however, the drained placenta has been regarded as having nofurther use and has typically been discarded. A major limitation of stemcell procurement from cord blood, moreover, has been the frequentlyinadequate volume of cord blood obtained, resulting in insufficient cellnumbers to effectively reconstitute bone marrow after transplantation.

Naughton et al. (U.S. Pat. No. 5,962,325 entitled “Three-dimensionalstromal tissue cultures” issued Oct. 5, 1999) discloses that fetalcells, including fibroblast-like cells and chondrocyte-progenitors, maybe obtained from umbilical cord or placenta tissue or umbilical cordblood.

Kraus et al. (U.S. Pat. No. 6,338,942, entitled “Selective expansion oftarget cell populations”, issued Jan. 15, 2002) discloses that apredetermined target population of cells may be selectively expanded byintroducing a starting sample of cells from cord blood or peripheralblood into a growth medium, causing cells of the target cell populationto divide, and contacting the cells in the growth medium with aselection element comprising binding molecules with specific affinity(such as a monoclonal antibody for CD34) for a predetermined populationof cells (such as CD34 cells), so as to select cells of thepredetermined target population from other cells in the growth medium.

Rodgers et al (U.S. Pat. No. 6,335,195 entitled “Method for promotinghematopoietic and mesenchymal cell proliferation and differentiation,”issued Jan. 1, 2002) discloses methods for ex vivo culture ofhematopoietic and mesenchymal stem cells and the induction oflineage-specific cell proliferation and differentiation by growth in thepresence of angiotensinogen, angiotensin I (AI), AI analogues, AIfragments and analogues thereof, angiotensin II (AII), AII analogues,AII fragments or analogues thereof or All AT₂ type 2 receptor agonists,either alone or in combination with other growth factors and cytokines.The stem cells are derived from bone marrow, peripheral blood orumbilical cord blood. The drawback of such methods, however, is thatsuch ex vivo methods for inducing proliferation and differentiation ofstem cells are time-consuming, as discussed above, and also result inlow yields of stem cells.

Because of restrictions on the collection and use of stem cells, and theinadequate numbers of cells typically collected from cord blood, stemcells are in critically short supply. Stem cells have the potential tobe used in the treatment of a wide variety of disorders, includingmalignancies, inborn errors of metabolism, hemoglobinopathies, andimmunodeficiencies. There is a critical need for a readily accessiblesource of large numbers of human stem cells for a variety of therapeuticand other medically related purposes. The present invention addressesthat need and others.

Additionally, there remains a need for the treatment of neurologicalconditions such as amylotrophic lateral sclerosis (ALS). Although recentstudies using irradiated mouse models of familial ALS, a less-commonform of ALS, have suggested that cord blood may be useful in thetreatment of this disease, the source issue discussed above makes thisoption less than ideal. See Ende et al., Life Sci. 67:53059 (2000).Thus, there remains a need for stem or progenitor cell populations thatcan be used to treat diseases, particularly larger amounts of thesepopulations when diseases such as ALS are being treated.

3. SUMMARY OF THE INVENTION

The present invention relates to cord blood compositions or stem orprogenitor cells therefrom in which said compositions are supplementedwith or contacted with embryonic-like stem cells that originate from apost-partum placenta. The embryonic-like stem cells which are thesubject of other applications can be used herein as a composition or amixture with other stem or progenitor cell populations. In accordancewith the present invention, the embryonic-like stem cells may contactedwith other stem or progenitor cell populations, including but notlimited to, umbilical cord blood, fetal and neonatal hematopoietic stemcells and progenitor cells, human stem cells and progenitor cellsderived from bone marrow. The embryonic-like stem cells and the mixedpopulations of embryonic-like stem cells and stem or progenitor cellshave a multitude of uses and applications, including but not limited to,therapeutic uses for transplantation and treatment and prevention ofdisease, and diagnostic and research uses.

In accordance with the present invention, populations of stem cells aremixed with populations of embryonic-like stem cells in order tosupplement, augment or enhance the concentrations of pluripotent andmultipotent stem cells in the stem cell populations. for example, in oneembodiment, umbilical cord blood, or stem or progenitor cells therefrom,is augmented or contacted with the embryonic-like stem cells of theinvention prior to administration to the patient. It is recognized thatthe embryonic-like stem cells may also be administered simultaneously orsequentially with the umbilical cord blood, or cells therefrom, However,contacting the cells of each before administration is preferred.

The embryonic-like stem cells of the invention may be characterized bythe presence of the following cell surface markers: CD10, CD29, CD44,CD54, CD90, SH2, SH3, SH4, OCT-4 and ABC-p, and the absence of thefollowing cell surface markers: CD34, CD38, CD45, SSEA3 and SSEA4. In apreferred embodiment, such embryonic-like stem cells may becharacterized by the presence of cell surface markers OCT-4 and APC-p.Embryonic-like stem cells originating from placenta have characteristicsof embryonic stem cells but are not derived from the embryo. In otherwords, the invention encompasses mixtures of cord blood andembryonic-like stem cells isolated from a placenta that are OCT-4+and/or ABC-p+. Such embryonic-like stem cells are as versatile (e.g.,pluripotent) as human embryonic stem cells.

In accordance with the present invention, populations of stem cells aremixed with embryonic-like stem cells that are pluripotent ormultipotent. Such embryonic-like stem cells can be isolated from theperfused placenta at different time points e.g., CD34+/CD38+,CD34+/CD38−, and CD34−/CD38− hematopoietic cells. In one embodiment,such cells may be used to supplement populations of hematopoictic stemcells, such as those found in umbilical cord blood, according to themethods of the invention.

The invention also provides a composition in which a mixture of stemcells with embryonic-like stem cells is contained within one bag orcontainer. In a preferred embodiment, the composition is apharmaceutically acceptable unit dose composition. In anotherembodiment, the invention provides a composition in which a populationof stem cells and a population of embryonic-like stem cells arecontained within two separate bags or containers. In certainembodiments, such a “two bag” kit may be mixed prior, in particularimmediately prior to, or at the time of administration to a patient inneed thereof. In other embodiments, the contents of each bag may beadministered separately to a patient, wherein the mixing of the two cellpopulations occurs in vivo. In other embodiments, the container issealed, air tight, and sterile.

The present invention relates to populations of stem cells are mixedwith embryonic-like stem cells. In accordance with the presentinvention, stems cells that may be mixed with embryonic-like stem cellsinclude, but are not limited to, umbilical cord blood, fetal andneonatal hematopoietic stem cells and progenitor cells, human stem cellsand progenitor cells derived from bone marrow. In a preferred embodimentof the present invention, the embryonic-like stem cells of the inventionare mixed with umbilical cord blood.

The present invention also provides methods of treating a patient inneed thereof by administration of a population of stem cellssupplemented with embryonic-like stem cells. In one embodiment, thesupplementation of the population of cord blood cells withembryonic-like stem cells occurs by mixing the stem cells andembryonic-like stem cells prior to administration of the combined or“spiked” population to the patient. In another embodiment, thesupplementation of the population of stem cells with embryonic-like stemcells occurs upon administration of the supplemented population to thepatient, e.g., by simultaneous administration of the cord blood cellsand the embryonic-like stem cells. In another embodiment, thesupplementation of the population of stem cells with embryonic-like stemcells occurs after administration of the cord blood cells to thepatient, e.g., by administering the embryonic-stem cells separatelyfrom, and before or after, administration of the stem cells.

According to the invention, populations of stem cells, e.g., umbilicalcord blood, supplemented with embryonic-like stem cells from theplacenta have a multitude of uses, including prophylactic, therapeuticand diagnostic uses. The supplemented populations of stem cells can beused for transplantation and/or to treat or prevent disease. In oneembodiment of the invention, the supplemented populations of cells areused to renovate and repopulate tissues and organs, thereby replacing orrepairing diseased tissues, organs or portions thereof. In anotherembodiment, the supplemented populations of stem cells can be used as adiagnostic to screen for genetic disorders or a predisposition for aparticular disease or disorder.

In another embodiment, the invention provides a method for isolatingother embryonic-like and/or multipotent or pluripotent stem cells froman extract or perfusate of a exsanguinated placenta and using them tosupplement populations of cord blood cells according to the methods ofthe invention.

The present invention also provides pharmaceutical compositions thatcomprise populations of stem cells, e.g., umbilical cord blood cells,that have been supplemented with one or more populations ofembryonic-like stem cells of the invention.

The present invention provides an isolated homogenous population ofhuman placental stem cells that has the potential to differentiate intoall cell types. In another embodiment, the population of human placentalstem cells has the potential to differentiate into one cell type. In yetanother embodiment, the population of human placental stem cells has thepotential to differentiate into several different cell types. Such cellsmay be used to supplement populations of stem cells, e.g., umbilicalcord blood, according to the methods of the invention.

The invention also encompasses pharmaceutical compositions that comprisepopulations of hematopoietic stem cells supplemented with one or morepopulations of cells that have high concentrations (or largerpopulations) of homogenous hematopoietic stem cells including, but notlimited to, CD34+/CD38− cells; CD34−/CD38− cells, and CD133⁺ cells. Oneor more of these cell populations can be used with, or mixed with,hematopoietic stem cells i.e., CD34+/CD38+ hematopoietic cells, obtainedfrom umbilical cord blood or other sources, for transplantation andother uses.

The present invention also provides methods of mixing a population ofstem, progenitor or cord blood cells, including banked or cryopreservedcord blood cells, with a population of embryonic-like stem cells. In oneembodiment, the two populations are physically mixed. In another aspectof this embodiment, the two populations are physically mixed and thentreated with a growth factor, e.g., a cytokine and/or an interleukin, toinduce cell differentiation. In another aspect of this embodiment, thestem cells and/or the embryonic-like stem cells are treated with agrowth factor, e.g., a cytokine and/or an interleukin, to induce celldifferentiation and then physically mixed. In one embodiment, the mixedpopulations are treated with a growth factor to induce differentiationinto a variety of cell types. In another embodiment, the mixedpopulations are treated with a growth factor to induce differentiationinto a particular cell type. In another embodiment, the mixedpopulations are treated with a growth factor to prevent or suppressdifferentiation into a particular cell type. In certain embodiments, theculture conditions can be controlled, e.g., the mixed population ofcells can be treated with a specific cocktail of cytokines orinterleukins to direct or induce differentiation to a specific celltype.

In another embodiment, the invention provides a method of treating apatient in need thereof comprising administration of a plurality ofumbilical cord blood cells and a plurality of embryonic-like stem cells.

In another embodiment, the invention provides a method of treatingmyelodysplasia which comprises administering umbilical cord blood cells(or stem cells isolated therefrom) and embryonic-like stem cells to apatient in need thereof. The invention also relates to new uses of humanplacental stem cells (embryonic-like stem cells). Methods of treating orpreventing disease with the compositions containing embryonic-like stemcells and other stem or progenitor cells or sources thereof are alsoencompassed herein. Similarly, methods of dosing such compositions areencompassed. finally, it should be noted that the compositions of theinvention can contain stem or progenitor cell populations from multipledonors. The invention includes the use of non-HLA matched compositionsin patients as well as HLA-matched compositions. blood type matchingwith the patient is preferred but not required when the compositionscontaining both embryonic-like stem cells and stem or progenitor cellsare used.

3.1. Definitions

As used herein, the term “bioreactor” refers to an ex vivo system forpropagating cells, producing or expressing biological materials andgrowing or culturing cells tissues, organoids, viruses, proteins,polynucleotides and microorganisms.

As used herein, the terms “cord blood” and “umbilical cord blood” areinterchangeable.

As used herein, the term “embryonic stem cell” refers to a cell that isderived from the inner cell mass of a blastocyst (e.g., a 4- to5-day-old human embryo) and that is pluripotent.

As used herein, the term “embryonic-like stem cell” refers to a cellthat is not derived from the inner cell mass of a blastocyst. As usedherein, an “embryonic-like stem cell” may also be referred to as a“placental stem cell,” preferably a human placental stem cell derivedfrom a post-partum perfused placenta. An embryonic-like stem cell ispreferably pluripotent. However, the stem cells which may be obtainedfrom the placenta include embryonic-like stem cells, multipotent cells,and committed progenitor cells. According to the methods of theinvention, embryonic-like stem cells derived from the placenta may becollected from the isolated placenta once it has been exsanguinated andperfused for a period of time sufficient to remove residual cells.

As used herein, the term “exsanguinated” or “exsanguination,” when usedwith respect to the placenta, refers to the removal and/or draining ofsubstantially all cord blood from the placenta. In accordance with thepresent invention, exsanguination of the placenta can be achieved by,for example, but not by way of limitation, draining, gravity inducedefflux, massaging, squeezing, pumping, etc. In a preferred embodiment,exsanguination of the placenta may further be achieved by perfusing,rinsing or flushing the placenta with a fluid that may or may notcontain agents, such as anticoagulants, to aid in the exsanguination ofthe placenta.

As used herein, the term to “mix” means to combine or blend into onemass or mixture; to put together into one mass so that the constituentparts are more or less homogeneous; to create or form by combiningingredients; to form by admixture, augmentation, supplementation, orcommingling; or to add an ingredient or element to another ingredient orelement, and vice-versa.

As used herein, the term “perfuse” or “perfusion” refers to the act ofpouring or passaging a fluid over or through an organ or tissue,preferably the passage of fluid through an organ or tissue withsufficient force or pressure to remove any residual cells, e.g.,non-attached cells from the organ or tissue. As used herein, the term“perfusate” refers to the fluid collected following its passage throughan organ or tissue. In a preferred embodiment, the perfusate containsone or more anticoagulants.

As used herein, the term “exogenous cell” refers to a “foreign” cell,i.e., a heterologous cell (i.e., a “non-self” cell derived from a sourceother than the placental donor) or autologous cell (i.e., a “self” cellderived from the placental donor) that is-derived from an organ ortissue other than the placenta.

As used herein, the term “organoid” refers to an aggregation of one ormore cell types assembled in superficial appearance or in actualstructure as any organ or gland of a mammalian body, preferably thehuman body.

As used herein, the term “multipotent cell” refers to a cell that hasthe capacity to grow into any of subset of the mammalian body'sapproximately 260 cell types. Unlike a pluripotent cell, a multipotentcell does not have the capacity to form all of the cell types.

As used herein, the term “pluripotent cell” refers to a cell that hascomplete differentiation versatility, i.e., the capacity to grow intoany of the mammalian body's approximately 260 cell types. A pluripotentcell can be self-renewing, and can remain dormant or quiescent within atissue. Unlike a totipotent cell (e.g., a fertilized, diploid egg cell),an embryonic stem cell cannot usually form a new blastocyst.

As used herein, the term “progenitor cell” refers to a cell that iscommitted to differentiate into a specific type of cell or to form aspecific type of tissue.

As used herein, the term “stem cell” refers to a master cell that canreproduce indefinitely to form the specialized cells of tissues andorgans. A stem cell is a developmentally pluripotent or multipotentcell. A stem cell can divide to produce two daughter stem cells, or onedaughter stem cell and one progenitor (“transit”) cell, which thenproliferates into the tissue's mature, fully formed cells. The “stemcell” used herein includes “progenitor cells” unless otherwise noted.

As used herein, the term “totipotent cell” refers to a cell that is ableto form a complete embryo (e.g., a blastocyst).

4. DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the unexpected discovery thatembryonic-like stem cells produced by the exsanguinated, perfused and/orcultured placenta are pluripotent stem cells that can be readilydifferentiated into any desired cell type. These embryonic-like stemcells can be used to supplement, augment or enhance populations of stemcells, including, but not limited to umbilical cord blood, fetal andneonatal hematopoietic stem cells and progenitor cells, human stem cellsand progenitor cells derived from bone marrow. In accordance with thepresent invention, populations of stem cells are mixed with populationsof embryonic-like stem cells in order to supplement, augment or enhancethe concentrations of pluripotent and multipotent stem cells in the stemcell populations. In accordance with the present invention, thepopulations of stem cells mixed with populations of embryonic-like stemcells have a multitude of uses and applications, including but notlimited to, therapeutic uses for transplantation and treatment andprevention of disease, and diagnostic and research uses.

The invention also provides a composition in which a mixture of stemcells and embryonic-like stem cells is contained within one bag orcontainer. In another embodiment, the invention provides a compositionin which a population of stem cells and a population of embryonic-likestem cells are contained within two separate bags or containers. Incertain embodiments, such a “two bag” composition may be mixed prior, inparticular immediately prior, to or at the time of administration to apatient in need thereof. In other embodiments, the contents of each bagmay be administered separately to a patient, wherein two cellpopulations are used adjunctively in vivo.

The present invention also provides methods of mixing a population ofstem or progenitor cells or cord blood including banked or cryopreservedcord blood with a population of embryonic-like stem cells. In oneembodiment, the two populations are physically mixed. In another aspectof this embodiment, the two populations are physically mixed and thentreated with a growth factor, e.g., a cytokine and/or an interleukin, toinduce cell differentiation. In another aspect of this embodiment, thestem cells and/or the embryonic-like stem cells are treated with agrowth factor, e.g., a cytokine and/or an interleukin, to induce celldifferentiation and then physically mixed.

The present invention also provides methods of mixing a population ofcommitted cells, e.g., a population of cells committed to differentiateinto neurons, muscle cells, hematopoietic, vascular cells, adipocytes,chondrocytes, osteocytes, hepatocytes, pancreatic, or cardiac cells,with a population of embryonic-like stem cells. In one embodiment, thetwo populations are physically mixed. In another aspect of thisembodiment, the two populations are physically mixed and then treatedwith a growth factor, e.g., a cytokine and/or an interleukin, to inducecell differentiation. In another aspect of this embodiment, thecommitted cells and/or the embryonic-like stem cells are treated with agrowth factor, e.g., a cytokine and/or an interleukin, to induce celldifferentiation and then physically mixed.

According to the methods of the invention, embryonic-like stem cells areextracted from a drained placenta by means of a perfusion technique thatutilizes either or both of the umbilical artery and the umbilical vein.The placenta is preferably drained by exsanguination and collection ofresidual blood (e.g., residual umbilical cord blood). The drainedplacenta is then processed in such a manner as to establish the ex vivo,natural bioreactor environment in which the resident embryonic-like stemcells within the parenchyma and extravascular space are recruited. Theembryonic-like stem cells migrate into the drained, emptymicrocirculation where, according to the methods of the invention, theyare collected, preferable by washing into a collecting vessel byperfusion.

As disclosed above, a number of different pluripotent or multipotentstem cells can be isolated from the perfused placenta at different timepoints during the perfusion, e.g., CD34+/CD38+, CD34+/CD38−, andCD34−/CD38− hematopoietic cells. In one embodiment, such cells may beused to supplement populations of stem cells, e.g., cord blood cells,according to the methods of the invention.

The present invention further provides an isolated homogenous populationof human placental stem cells that has the potential to differentiateinto all cell types. In another embodiment, the population of humanplacental stem cells has the potential to differentiate into one celltype. In yet another embodiment, the population of human placental stemcells has the potential to differentiate into several different celltypes. Such cells may be used to supplement populations of stem cells,e.g., cord blood cells, according to the methods of the invention.

The present invention also provides methods of mixing a population ofstem cells with a population of embryonic-like stem cells. In oneembodiment, the two populations are physically mixed. In another aspectof this embodiment, the two populations are physically mixed and thentreated with a growth factor, e.g., a cytokine and/or an interleukin, toinduce cell differentiation. In another aspect of this embodiment, thestem cells and/or the embryonic-like stem cells are treated with agrowth factor, e.g., a cytokine and/or an interleukin, to induce celldifferentiation and then physically mixed. In one embodiment, the mixedpopulations are treated with a growth factor to induce differentiationinto a variety of cell types. In another embodiment, the mixedpopulations are treated with a growth factor to induce differentiationinto a particular cell type. In another embodiment, the mixedpopulations are treated with a growth factor to prevent or suppressdifferentiation into a particular cell type. In certain embodiments, theculture conditions can be controlled, e.g., the mixed population ofcells can be treated with a specific cocktail of cytokines orinterleukins to direct or induce differentiation to a specific celltype.

The present invention provides pharmaceutical compositions that comprisepopulations of stem cells, e.g., cord blood cells, that have beensupplemented with one or more populations of embryonic-like stem cellsof the invention.

The invention also encompasses pharmaceutical compositions that comprisepopulations of stem cells, e.g., cord blood cells, supplemented with oneor more populations of cells that have high concentrations (or largerpopulations) of homogenous hematopoietic stem cells including, but notlimited to, CD34+/CD38− cells; and CD34−/CD38− cells. One or more ofthese cell populations can be used with, or mixed with, cord bloodhematopoictic cells, i.e., CD34+/CD38+ hematopoietic cells fortransplantation and other uses.

According to the invention, populations of stem cells, e.g., umbilicalcord blood, supplemented with embryonic-like stem cells from theplacenta have a multitude of uses, including therapeutic and diagnosticuses. The supplemented populations of stem cells can be used fortransplantation or to treat or prevent disease. In one embodiment of theinvention, the supplemented populations of cells are used to renovateand repopulate tissues and organs, thereby replacing or repairingdiseased tissues, organs or portions thereof. In another embodiment, thesupplemented populations of stem cells can be used as a diagnostic toscreen for genetic disorders or a predisposition for a particulardisease or disorder.

The present invention also provides methods of treating a patient inneed thereof by administration of a population of stem cellssupplemented with embryonic-like stem cells. In one embodiment, thesupplementation of the population of cord blood cells withembryonic-like stem cells occurs by mixing the stem cells andembryonic-like stem cells prior to administration of the supplementedpopulation to the patient. In another embodiment, the supplementation ofthe population of stem cells with embryonic-like stem cells occurs uponadministration of the supplemented population to the patient, e.g., bysimultaneous administration of the cord blood cells and theembryonic-like stem cells. In another embodiment, the supplementation ofthe population of stem cells with embryonic-like stem cells occurs afteradministration of the cord blood cells to the patient, e.g., byadministering the embryonic-stem cells separately from, and before orafter, administration of the stem cells.

4.1. Methods of Isolating and Culturing Placenta

4.1.1. Pretreatment of Placenta

According to the methods of the invention, a human placenta is recoveredshortly after its expulsion after birth and, in certain embodiments, thecord blood in the placenta is recovered. In certain embodiments, theplacenta is subjected to a conventional cord blood recovery process.Such cord blood recovery may be obtained commercially, e.g., LifeBankInc., Cedar Knolls, N.J., ViaCord, Cord Blood Registry and Cryocell. Thecord blood can be drained shortly after expulsion of the placenta.

In other embodiments, the placenta is pretreated according to themethods disclosed in co-pending application Ser. No. 10/076,180, filedFeb. 13, 2002, which is incorporated herein by reference in itsentirety.

4.1.2. Exsanguination of Placenta and Removal of Residual Cells

As disclosed in PCT publication WO 02/064755, published Aug. 22, 2002,which is incorporated herein by reference in its entirety, the placentaafter birth contains quiescent cells that can be activated if theplacenta is properly processed after birth. For example, after expulsionfrom the womb, the placenta is exsanguinated as quickly as possible toprevent or minimize apoptosis. Subsequently, as soon as possible afterexsanguination the placenta is perfused to remove blood, residual cells,proteins, factors and any other materials present in the organ.Materials debris may also be removed from the placenta. Perfusion isnormally continued with an appropriate perfusate for at least two tomore than twenty-four hours. The placenta can therefore readily be usedas a rich and abundant source of embryonic-like stem cells, which cellscan be used for research, including drug discovery, treatment andprevention of diseases, in particular transplantation surgeries ortherapies, and the generation of committed cells, tissues and organoids.

Further, surprisingly and unexpectedly, the human placental stem cellsproduced by the exsanguinated, perfused and/or cultured placenta arepluripotent stem cells that can readily be differentiated into anydesired cell type.

According to the methods of the invention, stem or progenitor cells,including, but not limited to embryonic-like stem cells, may berecovered from a placenta that is exsanguinated, i.e., completelydrained of the cord blood remaining after birth and/or a conventionalcord blood recovery procedure. According to the methods of theinvention, the methods for exsanguination of the placenta and removal ofresidual cells may be accomplished using any method known in the art,e.g., the methods disclosed in PCT publication WO 02/064755, publishedAug. 22, 2002, which is incorporated herein by reference in itsentirety.

4.1.3. Culturing Placenta

After exsanguination and a sufficient time of perfusion of the placenta,the embryonic-like stem cells are observed to migrate into theexsanguinated and perfused microcirculation of the placenta where,according to the methods of the invention, they are collected,preferably by washing into a collecting vessel by perfusion. In otherembodiments, the placenta is cultured, and the cells propagated aremonitored, sorted and/or characterized according to the methodsdescribed in PCT publication WO 02/064755, published Aug. 22, 2002,which is incorporated herein by reference in its entirety.

4.2. Collection of Cells from the Placenta

After exsanguination and perfusion of the placenta, embryonic-like stemcells migrate into the drained, empty microcirculation of the placentawhere, according to the invention, they are collected, preferably bycollecting the effluent perfusate in a collecting vessel.

In preferred embodiments, cells cultured in the placenta are isolatedfrom the effluent perfusate using techniques known by those skilled inthe art, such as, for example, density gradient centrifugation, magnetcell separation, flow cytometry, or other cell separation or sortingmethods well known in the art, and sorted.

In a specific embodiment, the embryonic-like stem cells are collectedfrom the placenta and, in certain embodiments, preserved, according tothe methods described in PCT publication WO 02/064755, published Aug.22, 2002, which is incorporated herein by reference in its entirety.

4.3. Embryonic-Like Stem Cells

Embryonic-like stem cells obtained in accordance with the methods of theinvention may include pluripotent cells, i.e., cells that have completedifferentiation versatility, that are self-renewing, and can remaindormant or quiescent within tissue. The stem cells which may be obtainedfrom the placenta include embryonic-like stem cells, multipotent cells,committed progenitor cells, and fibroblastoid cells.

The first collection of blood from the placenta is referred to as cordblood which contains predominantly CD34+ and CD38+ hematopoieticprogenitor cells. Within the first twenty-four hours of post-partumperfusion, high concentrations of CD34+ and CD38− hematopoieticprogenitor cells may be isolated from the placenta, along with highconcentrations of CD34− and CD38+ hematopoietic progenitor cells. Afterabout twenty-four hours of perfusion, high concentrations of CD34− andCD38− cells can be isolated from the placenta along with theaforementioned cells. The isolated perfused placenta of the inventionprovides a source of large quantities of stem cells enriched for CD34+and CD38− stem cells and CD34− and CD38+ stem cells. The isolatedplacenta which has been perfused for twenty-four hours or more providesa source of large quantities of stem cells enriched for CD34− and CD38−stem cells.

In a preferred embodiment, embryonic-like stem cells obtained by themethods of the invention are viable, quiescent, pluripotent stem cellsthat exist within a full-term human placenta and that can be recoveredfollowing successful birth and placental expulsion, resulting in therecovery of as many as one billion nucleated cells, which yield 50-100million multipotent and pluripotent stem cells.

The human placental stem cells provided by the placenta are surprisinglyembryonic-like, for example, the presence of the following cell surfacemarkers have been identified for these cells: SSEA3−, SSEA4−, OCT-4+ andABC-p⁺. Preferably, the embryonic-like stem cells of the invention arecharacterized by the presence of OCT-4+ and ABC-p⁺ cell surface markers.Thus, the invention encompasses stem cells which have not been isolatedor otherwise obtained from an embryonic source but which can beidentified by the following markers: SSAE3−, SSAE4−, OCT-4+ and ABC-p⁺.In one embodiment, the human placental stem cells do not express MHCClass 2 antigens.

The stem cells isolated from the placenta are homogenous, and sterile.Further, the stem cells are readily obtained in a form suitable foradministration to humans, i.e., they are of pharmaceutical grade.

Preferred embryonic-like stem cells obtained by the methods of theinvention may be identified by the presence of the following cellsurface markers: OCT-4+ and ABC-pt. Further, the invention encompassesembryonic stem cells having the following markers: CD10+, CD38−, CD29+,CD34−, CD44+, CD45−, CD54+, CD90+, SH2+, SH3+, SH4+, SSEA3−, SSEA4−,OCT-4+, and ABC-p⁺. Such cell surface markers are routinely determinedaccording to methods well known in the art, e.g. by flow cytometry,followed by washing and staining with an anti-cell surface markerantibody. For example, to determine the presence of CD-34 or CD-38,cells may be washed in PBS and then double-stained with anti-CD34phycoerythrin and anti-CD38 fluorescein isothiocyanate (BectonDickinson, Mountain View, Calif.).

In another embodiment, cells cultured in the placenta bioreactor areidentified and characterized by a colony forming unit assay, which iscommonly known in the art, such as Mesen Cult™ medium (stem cellTechnologies, Inc., Vancouver British Columbia)

The embryonic-like stem cells obtained by the methods of the inventionmay be induced to differentiate along specific cell lineages, includingadipogenic, chondrogenic, osteogenic, hematopoietic, myogenic,vasogenic, neurogenic, and hepatogenic. In certain embodiments,embryonic-like stem cells obtained according to the methods of theinvention are induced to differentiate for use in transplantation and exvivo treatment protocols. In certain embodiments, embryonic-like stemcells obtained by the methods of the invention are induced todifferentiate into a particular cell type and genetically engineered toprovide a therapeutic gene product. In a specific embodiment,embryonic-like stem cells obtained by the methods of the invention areincubated with a compound in vitro that induces it to differentiate,followed by direct transplantation of the differentiated cells to asubject. Thus, the invention encompasses methods of differentiating thehuman placental stem cells using standard culturing media. Further, theinvention encompasses hematopoietic cells, neuron cells, fibroblastcells, strand cells, mesenchymal cells and hepatic cells.

Embryonic-like stem cells may also be further cultured after collectionfrom the placenta using methods well known in the art, for example, byculturing on feeder cells, such as irradiated fibroblasts, obtained fromthe same placenta as the embryonic-like stem cells or from other humanor nonhuman sources, or in conditioned media obtained from cultures ofsuch feeder cells, in order to obtain continued long-term cultures ofembryonic-like stem cells. The embryonic-like stem cells may also beexpanded, either within the placenta before collection from theplacental bioreactor or in vitro after recovery from the placenta. Incertain embodiments, the embryonic-like stem cells to be expanded areexposed to, or cultured in the presence of, an agent that suppressescellular differentiation. Such agents are well-known in the art andinclude, but are not limited to, human Delta-1 and human Serrate-1polypeptides (see, Sakano et al., U.S. Pat. No. 6,337,387 entitled“Differentiation-suppressive polypeptide”, issued Jan. 8, 2002),leukemia inhibitory factor (LIF) and stem cell factor. Methods for theexpansion of cell populations are also known in the art (see e.g.,Emerson et al., U.S. Pat. No. 6,326,198 entitled “Methods andcompositions for the ex vivo replication of stem cells, for theoptimization of hematopoietic progenitor cell cultures, and forincreasing the metabolism, GM-CSF secretion and/or IL-6 secretion ofhuman stromal cells”, issued Dec. 4, 2001; Kraus et al., U.S. Pat. No.6,338,942, entitled “Selective expansion of target cell populations”,issued Jan. 15, 2002).

The embryonic-like stem cells may be assessed for viability,proliferation potential, and longevity using standard techniques knownin the art, such as trypan blue exclusion assay, fluorescein diacetateuptake assay, propidium iodide uptake assay (to assess viability); andthymidine uptake assay, MTT cell proliferation assay (to assessproliferation). Longevity may be determined by methods well known in theart, such as by determining the maximum number of population doubling inan extended culture.

In certain embodiments, the differentiation of stem cells or progenitorcells that are cultivated in the exsanguinated, perfused and/or culturedplacenta is modulated using an agent or pharmaceutical compositionscomprising a dose and/or doses effective upon single or multipleadministration, to exert an effect sufficient to inhibit, modulateand/or regulate the differentiation of a cell collected from theplacenta.

Agents that can induce stem or progenitor cell differentiation are wellknown in the art and include, but are not limited to, Ca²⁺, EGF, α-FGF,β-FGF, PDGF, keratinocyte growth factor (KGF), TGF-β, cytokines (e.g.,IL-1α, IL-1β, IFN-γ, TFN), retinoic acid, transferrin, hormones (e.g.,androgen, estrogen, insulin, prolactin, triiodothyronine,hydrocortisone, dexamethasone), sodium butyrate, TPA, DMSO, NMF, DMF,matrix elements (e.g., collagen, laminin, heparan sulfate, Matrigel™),or combinations thereof.

Agents that suppress cellular differentiation are also well-known in theart and include, but are not limited to, human Delta-1 and humanSerrate-1 polypeptides (see, Sakano et al., U.S. Pat. No. 6,337,387entitled “Differentiation-suppressive polypeptide”, issued Jan. 8,2002), leukemia inhibitory factor (LIF), and stem cell factor.

The agent used to modulate differentiation can be introduced into theplacental bioreactor to induce differentiation of the cells beingcultured in the placenta. Alternatively, the agent can be used tomodulate differentiation in vitro after the cells have been collected orremoved from the placenta.

Determination that a stem cell has differentiated into a particular celltype may be accomplished by methods well-known in the art, e.g.,measuring changes in morphology and cell surface markers usingtechniques such as flow cytometry or immunocytochemistry (e.g., stainingcells with tissue-specific or cell-marker specific antibodies), byexamination of the morphology of cells using light or confocalmicroscopy, or by measuring changes in gene expression using techniqueswell known in the art, such as PCR and gene-expression profiling.

4.4. Supplementing Populations of Stem Cells with Embryonic-Like StemCells

The present invention relates to populations of stem cells are mixedwith embryonic-like stem cells. In accordance with the presentinvention, stems cells that may be mixed with embryonic-like stem cellsinclude, but are not limited to, umbilical cord blood, fetal andneonatal hematopoietic stem cells and progenitor cells, human stem cellsand progenitor cells derived from bone marrow. In a preferred embodimentof the present invention, the embryonic-like stem cells of the inventionare mixed with umbilical cord blood.

The present invention provides an isolated homogenous population ofhuman placental stem cells (embryonic-like stem cells) which has thepotential to differentiate into all cell types. Such cells may be usedto supplement populations of stem cells, e.g., cord blood cells,according to the methods of the invention.

The invention also provides populations of cord blood cells that havebeen supplemented (i.e., mixed, combined or augmented) with populationsof embryonic-like stem cells that originate from a placenta.

The supplemented populations are very versatile, in that they containpopulations of cells that are pluripotent or multipotent stem cells,e.g., cells displaying a CD34+/CD38+, CD34+/CD38− or CD34−/CD38−phenotype.

In accordance with the present invention the supplemented populations ofstem cells of the invention contain embryonic-like stem cells and otherstem or progenitor cells at a ratio of 100,000,000:1, 50,000,000:1,20,000,000:1, 10,000,000:1, 5,000,000:1, 2,000,000:1, 1,000,000:1,500,000:1, 200,000:1, 100,000:1, 50,000:1, 20,000:1, 10,000:1, 5,000:1,2,000:1, 1,000:1, 500:1, 200:1, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1;1:2; 1:5; 1:10; 1:100; 1:200; 1:500; 1:1,000; 1:2,000; 1:5,000;1:10,000; 1:20,000; 1:50,000; 1:100,000; 1:500,000; 1:1,000,000;1:2,000,000; 1:5,000,000; 1:10,000,000; 1:20,000,000; 1:50,000,000; or1:100,000,000, comparing numbers of total nucleated cells in eachpopulation.

In another embodiment, the invention provides methods for supplementing,mixing, combining or augmenting stem cells, e.g., umbilical cord blood,with a composition of the invention, e.g., a population of pureembryonic-like placental stem cells or a population of cells enrichedfor embryonic-like placental stem cells. In one embodiment, an aliquot(or population) of embryonic-like placental stem cells is added to analiquot of umbilical cord blood. before delivery to a patient in needthereof.

The present invention also provides methods of supplementing apopulation of cord blood cells with a population of embryonic-like stemcells. In one embodiment, the two populations are physically mixed. Inanother aspect of this embodiment, the two populations are physicallymixed and then treated with a growth factor, e.g., a cytokine and/or aninterleukin, to induce cell differentiation. In another aspect of thisembodiment, the cord blood cells and/or the embryonic-like stem cellsare treated with a growth factor, e.g., a cytokine and/or aninterleukin, to induce cell differentiation and then physically mixed.

The present invention also provides methods of treating a patient inneed thereof by administration of a population of cord blood cellssupplemented with embryonic-like stem cells. In one embodiment, thesupplementing of the population of cord blood cells with embryonic-likestem cells occurs by mixing the cord blood cells and embryonic-like stemcells prior to administration of the supplemented population to thepatient. In another embodiment, supplementing the population of cordblood cells with embryonic-like stem cells occurs upon administration ofthe supplemented population to the patient, e.g., by simultaneousadministration of the cord blood cells and the embryonic-like stemcells. In another embodiment, supplementing of the population of cordblood cells with embryonic-like stem cells occurs after administrationof the cord blood cells to the patient, e.g., by administering theembryonic-stem cells separately from, and before or after,administration of the cord blood cells.

In one embodiment, the invention provides methods for supplementing cordblood cells with embryonic-like stem cells, wherein the mixture iscontained within one bag. In another embodiment, the invention providesmethods for supplementing cord blood cells with embryonic-like stemcells, wherein the cord blood cells and the embryonic-like stem cellsare each contained in a separate bags. Such a “two bag” composition maybe mixed prior to or at the time of administration to a patient in needthereof.

In another embodiment, an aliquot (or population) of embryonic-likeplacental stem cells are conditioned before being added to, and mixedinto, an aliquot of umbilical cord blood before delivery to a patient inneed thereof. For example, in one aspect of this embodiment, apopulation of embryonic-like placental stem cells is induced todifferentiate into a particular cell lineage, e.g., a hematopoietic,neuronal, adipogenic, chondrogenic, osteogenic, hepatogenic, pancreatic,or myogenic lineage, as disclosed above in Section 4.3, by exposure to,e.g., cytokines (e.g., IL-1α, IL-1β, IFN-γ, TFN), retinoic acid,transferrin, hormones (e.g., androgen, estrogen, insulin, prolactin,triiodothyronine, hydrocortisone, dexamethasone), sodium butyrate, TPA,DMSO, NMF, DMF, matrix elements (e.g., collagen, laminin, heparansulfate, Matrigel™), or combinations thereof, before being added to, andmixed into, an aliquot of umbilical cord blood.

In another aspect of this embodiment, a population of embryonic-likeplacental stem cells is conditioned by being exposed to an agent thatsuppresses differentiation, e.g., human Delta-1 and human Serrate-1polypeptides, or combinations thereof, before being added to, and mixedinto, an aliquot of umbilical cord blood.

In another embodiment, an aliquot (or population) of non-conditionedembryonic-like placental stem cells and an aliquot of umbilical cordblood are mixed, and the mixed population of cells is conditioned beforebeing delivery to a patient in need thereof. In specific embodiments,the mixed population of embryonic-like placental stem cells andumbilical cord blood cells are conditioned with an agent that induces orsuppresses cell differentiation as disclosed above.

In a specific embodiment, a population of embryonic-like stem cells ofthe invention is added to, or mixed into, a population of umbilical cordblood cells prior to administration to a patient in need thereof. Inanother specific embodiment, a population of embryonic-like stem cellsof the invention is added to, or mixed into, a population of umbilicalcord blood cells during, or simultaneous with, administration to apatient in need thereof. In another specific embodiment, a population ofembryonic-like stem cells of the invention and a population of umbilicalcord blood cells are administered sequentially to a patient in needthereof. In one embodiment, the population of embryonic-like stem cellsis administered first and the population of umbilical cord blood cellsis administered second. In another embodiment, the population ofumbilical cord blood cells is administered first and the population ofembryonic-like placental stem cells is administered second.

The populations of cord blood cells spiked with embryonic-like stemcells may be cultured, induced to propagate, and/or induced todifferentiate under a variety of conditions, including but not limitedto treating the spiked populations by introduction of nutrients,hormones, vitamins, growth factors, or any combination thereof, into theculture medium. Serum and other growth factors may be added to theculture medium. Growth factors are usually proteins and include, but arenot limited to: cytokines, lymphokines, interferons, colony stimulatingfactors (CSF's), interferons, chemokines, and interleukins. Other growthfactors that may be used include recombinant human hematopoictic growthfactors including ligands, stem cell factors, thrombopoeitin (Tpo),granulocyte colony-stimulating factor (G-CSF), leukemia inhibitoryfactor, basic fibroblast growth factor, placenta derived growth factorand epidermal growth factor. In one embodiment, the supplementedpopulations are treated with a growth factor to induce differentiationinto a variety of cell types. In another embodiment, the spikedpopulations are treated with a growth factor to induce differentiationinto a particular cell type. In another embodiment, the supplementedpopulations are treated with a growth factor to prevent or suppressdifferentiation into a particular cell type.

In certain embodiments of the invention, the methods of supplementing apopulation of cord blood comprise (a) induction of differentiation ofembryonic-like stem cells, (b) mixing the embryonic-like stem cells witha population of cord blood cells and (c) administration of the mixtureto a patient in need thereof.

In other embodiments of the invention, the methods of supplementing apopulation of cord blood comprise (a) mixing the embryonic-like stemcells with a population of cord blood cells; (b) induction ofdifferentiation of the mixture of the spiked population of cord bloodcells and embryonic-like stem cells and (c) administration of themixture to a patient in need thereof.

In other embodiments of the invention, the methods of supplementing apopulation of cord blood comprise (a) administration of a mixture ofcord blood cells supplemented with embryonic-like stem cells to apatient in need thereof and (b) induction of differentiation of themixture and (c) administration of the mixture to a patient in needthereof.

In certain embodiments, stem or progenitor cells are induced todifferentiate into a particular cell type, by exposure to a growthfactor, according to methods well known in the art. In specificembodiments, the growth factor is: GM-CSF, IL-4, Flt3L, CD40L,IFN-alpha, TNF-alpha, IFN-gamma, IL-2, IL-6, retinoic acid, basicfibroblast growth factor, TGF-beta-1, TGF-beta-3, hepatocyte growthfactor, epidermal growth factor, cardiotropin-1, angiotensinogen,angiotensin I (AI), angiotensin II (AII), AII AT₂ type 2 receptoragonists, or analogs or fragments thereof.

In one embodiment, stem or progenitor cells are induced to differentiateinto neurons, according to methods well known in the art, e.g., byexposure to β-mercaptoethanol or to DMSO/butylated hydroxyanisole,according to the methods disclosed in Section 5.4.1.

In another embodiment, stem or progenitor cells are induced todifferentiate into adipocytes, according to methods well known in theart, e.g., by exposure to dexamethasone, indomethacin, insulin and IBMX,according to the methods disclosed in Section 5.4.2.

In another embodiment, stem or progenitor cells are induced todifferentiate into chondrocytes, according to methods well known in theart, e.g., by exposure to TGF-.beta-3, according to the methodsdisclosed in Section 5.4.3.

In another embodiment, stem or progenitor cells are induced todifferentiate into osteocytes, according to methods well known in theart, e.g., by exposure to dexamethasone, ascorbic acid-2-phosphate andbeta-glycerophosphate, according to the methods disclosed in Section5.4.4.

In another embodiment, stem or progenitor cells are induced todifferentiate into hepatocytes, according to methods well known in theart, e.g., by exposure to IL-6+/−IL-15, according to the methodsdisclosed in Section 5.4.5.

In another embodiment, stem or progenitor cells are induced todifferentiate into pancreatic cells, according to methods well known inthe art, e.g., by exposure to basic fibroblast growth factor, andtransforming growth factor beta-1, according to the methods disclosed inSection 5.4.6.

In another embodiment, stem or progenitor cells are induced todifferentiate into cardiac cells, according to methods well known in theart, e.g., by exposure to retinoic acid, basic fibroblast growth factor,TGF-beta-1 and epidermal growth factor, by exposure to cardiotropin-1 orby exposure to human myocardium extract, according to the methodsdisclosed in Section 5.4.7.

In another embodiment, the embryonic-like stem cells are stimulated toproduce bioactive molecules, such as immunoglobulins, hormones, enzymes.

In another embodiment, the embryonic-like stem cells are stimulated toproliferate, for example, by administration of erythropoietin,cytokines, lymphokines, interferons, colony stimulating factors (CSF's),interferons, chemokines, interleukins, recombinant human hematopoieticgrowth factors including ligands, stem cell factors, thrombopoeitin(Tpo), interleukins, and granulocyte colony-stimulating factor (G-CSF)or other growth factors.

In another embodiment, the embryonic-like stem cells are geneticallyengineered either prior to, or after collection from, the placenta,using, for example, a viral vector such as an adenoviral or retroviralvector, or by using mechanical means such as liposomal or chemicalmediated uptake of the DNA.

A vector containing a transgene can be introduced into a cell ofinterest by methods well known in the art, e.g., transfection,transformation, transduction, electroporation, infection,microinjection, cell fusion, DEAE dextran, calcium phosphateprecipitation, liposomes, LIPOFECTIN™, lysosome fusion, syntheticcationic lipids, use of a gene gun or a DNA vector transporter, suchthat the transgene is transmitted to daughter cells, e.g., the daughterembryonic-like stem cells or progenitor cells produced by the divisionof an embryonic-like stem cell. For various techniques fortransformation or transfection of mammalian cells, see Keown et al.,1990, Methods Enzymol. 185: 527-37; Sambrook et al., 2001, MolecularCloning, A Laboratory Manual, Third Edition, Cold Spring HarborLaboratory Press, N.Y.

Preferably, the transgene is introduced using any technique, so long asit is not destructive to the cell's nuclear membrane or other existingcellular or genetic structures. In certain embodiments, the transgene isinserted into the nucleic genetic material by microinjection.Microinjection of cells and cellular structures is commonly known andpracticed in the art.

For stable transfection of cultured mammalian cells, such as theembryonic-like stem cells, only a small fraction of cells may integratethe foreign DNA into their genome. The efficiency of integration dependsupon the vector and transfection technique used. In order to identifyand select integrants, a gene that encodes a selectable marker (e.g.,for resistance to antibiotics) is generally introduced into the hostembryonic-like stem cell along with the gene sequence of interest.Preferred selectable markers include those that confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die). Such methods are particularlyuseful in methods involving homologous recombination in mammalian cells(e.g., in embryonic-like stem cells) prior to introduction ortransplantation of the recombinant cells into a subject or patient.

A number of selection systems may be used to select transformed hostembryonic-like cells. In particular, the vector may contain certaindetectable or selectable markers. Other methods of selection include butare not limited to selecting for another marker such as: the herpessimplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223),hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski,1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77: 3567; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78: 2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30: 147).

The transgene may integrate into the genome of the cell of interest,preferably by random integration. In other embodiments the transgene mayintegrate by a directed method, e.g., by directed homologousrecombination (i.e., “knock-in” or “knock-out” of a gene of interest inthe genome of cell of interest), Chappel, U.S. Pat. No. 5,272,071; andPCT publication No. WO 91/06667, published May 16, 1991; U.S. Pat. No.5,464,764; Capecchi et al., issued Nov. 7, 1995; U.S. Pat. No.5,627,059, Capecchi et al. issued, May 6, 1997; U.S. Pat. No. 5,487,992,Capecchi et al., issued Jan. 30, 1996).

Methods for generating cells having targeted gene modifications throughhomologous recombination are known in the art. The construct willcomprise at least a portion of a gene of interest with a desired geneticmodification, and will include regions of homology to the target locus,i.e., the endogenous copy of the targeted gene in the host's genome. DNAconstructs for random integration, in contrast to those used forhomologous recombination, need not include regions of homology tomediate recombination. Markers can be included in the targetingconstruct or random construct for performing positive and negativeselection for insertion of the transgene.

To create a homologous recombinant cell, e.g., a homologous recombinantembryonic-like stem cell, endogenous placental cell or exogenous cellcultured in the placenta, a homologous recombination vector is preparedin which a gene of interest is flanked at its 5′ and 3′ ends by genesequences that are endogenous to the genome of the targeted cell, toallow for homologous recombination to occur between the gene of interestcarried by the vector and the endogenous gene in the genome of thetargeted cell. The additional flanking nucleic acid sequences are ofsufficient length for successful homologous recombination with theendogenous gene in the genome of the targeted cell. Typically, severalkilobases of flanking DNA (both at the 5′ and 3′ ends) are included inthe vector. Methods for constructing homologous recombination vectorsand homologous recombinant animals from recombinant stem cells arecommonly known in the art (see, e.g., Thomas and Capecchi, 1987, Cell51: 503; Bradley, 1991, Curr. Opin. Bio/Technol. 2: 823-29; and PCTPublication Nos. WO 90/11354, WO 91/01140, and WO 93/04169.

In one embodiment, the genome of an exogenous cell cultured in theplacenta according to the methods of the invention is a target of genetargeting via homologous recombination or via random integration.

In a specific embodiment, the methods of Bonadio et al. (U.S. Pat. No.5,942,496, entitled Methods and compositions for multiple gene transferinto bone cells, issued Aug. 24, 1999; and PCT WO95/226 11, entitledMethods and compositions for stimulating bone cells, published Aug. 24,1995) are used to introduce nucleic acids into a cell of interest, suchas a stem cell, progenitor cell or exogenous cell cultured in theplacenta, e.g., bone progenitor cells.

4.5. Uses of Embryonic-Like Stem Cells and Supplemented Populations ofStem Cells

Embryonic-like stem cells may be obtained from perfused placentasaccording to the methods described in copending U.S. application Ser.No. 01/076,180, filed Feb. 13, 2002.

The placental stem cell (embryonic-like stem cell) may be induced todifferentiate into a particular cell type, either ex vivo or in vivo.For example, pluripotent embryonic-like stem cells may be injected intoa damaged organ, and for organ neogenesis and repair of injury in vivo.Such injury may be due to such conditions and disorders including, butnot limited to, myocardial infarction, seizure disorder, multiplesclerosis, stroke, hypotension, cardiac arrest, ischemia, inflammation,age-related loss of cognitive function, radiation damage, cerebralpalsy, neurodegenerative disease, Alzheimer's disease, Parkinson'sdisease, Leigh disease, AIDS dementia, memory loss, amyotrophic lateralsclerosis, ischemic renal disease, brain or spinal cord trauma,heart-lung bypass, glaucoma, retinal ischemia, or retinal trauma.

The embryonic-like stem cells isolated from the placenta, alone or incombination with stem or progenitor cell populations (i.e., the cellcompositions of the invention) may be used, in specific embodiments, inautologous or heterologous enzyme replacement therapy to treat specificdiseases or conditions, including, but not limited to lysosomal storagediseases, such as Tay-Sachs, Niemann-Pick, Fabry's, Gaucher's, Hunter's,and Hurler's syndromes, as well as other gangliosidoses,mucopolysaccharidoses, and glycogenoses.

In other embodiments, the embryonic-like stem cells, alone or incombination with stem or progenitor cell populations, may be used asautologous or heterologous transgene carriers in gene therapy to correctinborn errors of metabolism, adrenoleukodystrophy, cystic fibrosis,glycogen storage disease, hypothyroidism, sickle cell anemia, Pearsonsyndrome, Pompe's disease, phenylketonuria (PKU), porphyrias, maplesyrup urine disease, homocystinuria, mucoplysaccharide nosis, chronicgranulomatous disease and tyrosinemia and Tay-Sachs disease or to treatcancer, tumors or other pathological conditions.

In other embodiments, the cell compositions may be used in autologous orheterologous tissue regeneration or replacement therapies or protocols,including, but not limited to treatment of corneal epithelial defects,cartilage repair, facial dermabrasion, mucosal membranes, tympanicmembranes, intestinal linings, neurological structures (e.g., retina,auditory neurons in basilar membrane, olfactory neurons in olfactoryepithelium), burn and wound repair for traumatic injuries of the skin,or for reconstruction of other damaged or diseased organs or tissues.

The large numbers of embryonic-like stem cells and/or progenitorobtained using the methods of the invention would, in certainembodiments, reduce the need for large bone marrow donations.Approximately 1×10⁸ to 2×10⁸ bone marrow mononuclear cells per kilogramof patient weight must be infused for engraftment in a bone marrowtransplantation (i.e., about 70 ml of marrow for a 70 kg donor). Toobtain 70 ml requires an intensive donation and significant loss ofblood in the donation process. In a specific embodiment, cells from asmall bone marrow donation (e.g., 7-10 ml) could be expanded bypropagation in a placental bioreactor before infusion into a recipient.

Furthermore, a small number of stem cells and progenitor cells normallycirculate in the blood stream. In another embodiment, such exogenousstem cells or exogenous progenitor cells are collected by apheresis, aprocedure in which blood is withdrawn, one or more components areselectively removed, and the remainder of the blood is reinfused intothe donor. The exogenous cells recovered by apheresis are expanded bypropagation in a placental bioreactor, thus eliminating the need forbone marrow donation entirely.

While the blood cells regenerate between chemotherapy treatments,however, the cancer has time to grow and possibly become more resistantto the chemotherapy drugs due to natural selection. Therefore, thelonger chemotherapy is given and the shorter the duration betweentreatments, the greater the odds of successfully killing the cancer. Toshorten the time between chemotherapy treatments, embryonic-like stemcells or progenitor cells collected according to the methods of theinvention, alone or in combination with other stem cell or progenitorcell populations, could be introduced into the patient. Such treatmentwould reduce the time the patient would exhibit a low blood cell count,and would therefore permit earlier resumption of the chemotherapytreatment.

The embryonic-like stem cells, progenitor cells, foreign cells, orengineered cells obtained from a placenta according to the methods ofthe invention, alone or in combination with other stem cell orprogenitor cell populations, can be used in the manufacture of a tissueor organ in vivo. The methods of the invention encompass using cellsobtained from the placenta, e.g., embryonic-like stem cells, progenitorcells, or foreign stem or progenitor cells, to seed a matrix and to becultured under the appropriate conditions to allow the cells todifferentiate and populate the matrix. The tissues and organs obtainedby the methods of the invention may be used for a variety of purposes,including research and therapeutic purposes.

The embryonic-like stem cells and the supplemented populations of stemcells of the invention can also be used for a wide variety ofprophylactic or therapeutic protocols in which a tissue or organ of thebody is augmented, repaired or replaced by the engraftment,transplantation or infusion of a desired cell population, such as a stemcell or progenitor cell population. The embryonic-like stem cells andthe supplemented populations of stem cells of the invention can be usedto replace or augment existing tissues, to introduce new or alteredtissues, or to join together biological tissues or structures. Theembryonic-like stem and supplemented stem cell populations of theinvention can also be substituted for embryonic stem cells intherapeutic protocols described herein in which embryonic stem cellswould be typically be used.

In a preferred embodiment of the invention, embryonic-like stem cellsand supplemented stem cell populations may be used as autologous andallogenic, including matched and mismatched HLA type hematopoietictransplants. In accordance with the use of embryonic-like stem cells asallogenic hematopoietic transplants it may be necessary to treat thehost to reduce immunological rejection of the donor cells, such as thosedescribed in U.S. Pat. No. 5,800,539, issued Sep. 1, 1998; and U.S. Pat.No. 5,806,529, issued Sep. 15, 1998, both of which are incorporatedherein by reference.

For example, embryonic-like stem cells and supplemented stem cellpopulations of the invention can be used in therapeutic transplantationprotocols, e.g., to augment or replace stem or progenitor cells of theliver, pancreas, kidney, lung, nervous system, muscular system, bone,bone marrow, thymus, spleen, mucosal tissue, gonads, or hair.

Embryonic-like stem cells and supplemented stem cell populations may beused instead of specific classes of progenitor cells (e.g.,chondrocytes, hepatocytes, hematopoietic cells, pancreatic parenchymalcells, neuroblasts, muscle progenitor cells, etc.) in therapeutic orresearch protocols in which progenitor cells would typically be used.

Embryonic-like stem cells and supplemented stem cell populations of theinvention can be used for augmentation, repair or replacement ofcartilage, tendon, or ligaments. For example, in certain embodiments,prostheses (e.g., hip prostheses) are coated with replacement cartilagetissue constructs grown from embryonic-like stem cells of the invention.In other embodiments, joints (e.g., knee) are reconstructed withcartilage tissue constructs grown from embryonic-like stem cells.Cartilage tissue constructs can also be employed in major reconstructivesurgery for different types of joints (for protocols, see e.g., Resnick,D., and Niwayama, G., eds., 1988, Diagnosis of Bone and Joint Disorders,2d ed., W. B. Saunders Co.).

The embryonic-like stem cells and supplemented stem cell populations ofthe invention can be used to repair damage of tissues and organsresulting from trauma, metabolic disorders, or disease. In such anembodiment, a patient can be administered embryonic-like stem cells,alone or combined with other stem or progenitor cell populations, toregenerate or restore tissues or organs which have been damaged as aconsequence of disease, e.g., enhance immune system followingchemotherapy or radiation, repair heart tissue following myocardialinfarction.

The embryonic-like stem cells and supplemented stem cell populations ofthe invention can be used to augment or replace bone marrow cells inbone marrow transplantation. Human autologous and allogenic bone marrowtransplantation are currently used as therapies for diseases such asleukemia, lymphoma and other life-threatening disorders. The drawback ofthese procedures, however, is that a large amount of donor bone marrowmust be removed to insure that there is enough cells for engraftment.

The embryonic-like stem cells and supplemented stem cell populations ofthe invention can provide stem cells and progenitor cells that wouldreduce the need for large bone marrow donation. It would also be,according to the methods of the invention, to obtain a small marrowdonation and then expand the number of stem cells and progenitor cellsculturing and expanding in the placenta before infusion ortransplantation into a recipient.

The embryonic-like stem cells and supplemented stem cell populations ofthe invention may be used, in specific embodiments, in autologous orheterologous enzyme replacement therapy to treat specific diseases orconditions, including, but not limited to lysosomal storage diseases,such as Tay-Sachs, Niemann-Pick, Fabry's, Gaucher's, Hunter's, Hurler'ssyndromes, as well as other gangliosidoses, mucopolysaccharidoses, andglycogenoses.

In other embodiments, the cells may be used as autologous orheterologous transgene carriers in gene therapy to correct inborn errorsof metabolism such as adrenoleukodystrophy, cystic fibrosis, glycogenstorage disease, hypothyroidism, sickle cell anemia, Pearson syndrome,Pompe's disease, phenylketonuria (PKU), and Tay-Sachs disease,porphyrias, maple syrup urine disease, homocystinuria,mucopolypsaccharide nosis, chronic granulomatous disease, andtyrosinemia. or to treat cancer, tumors or other pathological orneoplastic conditions.

In other embodiments, the cells may be used in autologous orheterologous tissue regeneration or replacement therapies or protocols,including, but not limited to treatment of corneal epithelial defects,cartilage repair, facial dermabrasion, mucosal membranes, tympanicmembranes, intestinal linings, neurological structures (e.g., retina,auditory neurons in basilar membrane, olfactory neurons in olfactoryepithelium), burn and wound repair for traumatic injuries of the skin,scalp (hair) transplantation, or for reconstruction of other damaged ordiseased organs or tissues.

The large numbers of embryonic-like stem cells and/or progenitorobtained using the methods of the invention would, in certainembodiments, reduce the need for large bone marrow donations.Approximately 1×10⁸ to 2×10⁸ bone marrow mononuclear cells per kilogramof patient weight must be infused for engraftment in a bone marrowtransplantation (i.e., about 70 ml of marrow for a 70 kg donor). Toobtain 70 ml requires an intensive donation and significant loss ofblood in the donation process. In a specific embodiment, cells from asmall bone marrow donation (e.g., 7-10 ml) could be expanded bypropagation in a placental bioreactor before infusion into a recipient.

In another embodiment, the embryonic-like stem cells and supplementedstem cell populations of the invention can be used in a supplementaltreatment in addition to chemotherapy. Most chemotherapy agents used totarget and destroy cancer cells act by killing all proliferating cells,i.e., cells going through cell division. Since bone marrow is one of themost actively proliferating tissues in the body, hematopoictic stemcells are frequently damaged or destroyed by chemotherapy agents and inconsequence, blood cell production is diminishes or ceases. Chemotherapymust be terminated at intervals to allow the patient's hematopoieticsystem to replenish the blood cell supply before resuming chemotherapy.It may take a month or more for the formerly quiescent stem cells toproliferate and increase the white blood cell count to acceptable levelsso that chemotherapy may resume (when again, the bone marrow stem cellsare destroyed).

While the blood cells regenerate between chemotherapy treatments,however, the cancer has time to grow and possibly become more resistantto the chemotherapy drugs due to natural selection. Therefore, thelonger chemotherapy is given and the shorter the duration betweentreatments, the greater the odds of successfully killing the cancer. Toshorten the time between chemotherapy treatments, embryonic-like stemcells or progenitor cells collected according to the methods of theinvention could be introduced into the patient. Such treatment wouldreduce the time the patient would exhibit a low blood cell count, andwould therefore permit earlier resumption of the chemotherapy treatment.

In another embodiment, the human placental stem cells can be used totreat or prevent genetic diseases such as chronic granulomatous disease.

4.6. Pharmaceutical Compositions

The present invention encompasses pharmaceutical compositions whichcomprise the embryonic-like stem cells and supplemented stem cellpopulations of the invention. The present invention encompassespharmaceutical compositions comprising a dose and/or doses effectiveupon single or multiple administration, prior to or followingtransplantation of conditioned or unconditioned human progenitor stemcells, that are able to exert an effect sufficient to inhibit, modulateand/or regulate the differentiation of human pluripotent and multipotentprogenitor stem cells of placental origin into one or more celllineages, for example, mesodermal, adipose, chondrocytic, osteocytic,myocytic, vascular, neural, endothelial, hepatic, kidney, pancreatic,and/or hematopoietic lineage cells.

In accordance with this embodiment, the embryonic-like stem cells andsupplemented stem cell populations of the invention may be formulated asan injectable (e.g., PCT WO 96/39101, incorporated herein by referencein its entirety). In an alternative embodiment, the cells and tissues ofthe present invention may be formulated using polymerizable or crosslinking hydrogels as described in U.S. Pat. Nos. 5,709,854; 5,516,532;5,654,381; each of which is incorporated by reference in their entirety.The embryonic-like stem cells may be administered as obtained from theplacenta, or may be spiked into umbilical cord blood and administered asa mixed cell composition, or may be placed into anyphysiologically-acceptable buffer or fluid for administration to anindividual.

The invention also encompasses pharmaceutical compositions that havehigh concentrations (or larger populations) of homogenous embryonic-likestem cells, wherein one or more of these cell populations can be usedwith, or as a mixture with, other stem or progenitor cells, for use intransplantation and other uses. Other stem or progenitor cells mayinclude but are not limited to: adipogenic, chondrogenic, osteogenic,hematopoietic, myogenic, vasogenic, neurogenic, and hepatogenic stemcells; mesenchymal stem cells, stromal cells, endothelial cells,hepatocytes, keratinocytes, and stem or progenitor cells for aparticular cell type, tissue or organ, including but not limited toneurons, myelin, muscle, blood, bone marrow, skin, heart, connectivetissue, lung, kidney, liver, and pancreas (e.g., pancreatic isletcells).

In one embodiment, the invention provides pharmaceutical compositionsthat have high concentrations (or larger populations) of homogenoushematopoietic stem cells including but not limited to CD34+/CD38− cells;and CD34−/CD38− cells. One or more of these cell populations can be usedwith, or as a mixture with, other stem cells, for use in transplantationand other uses. In a specific embodiment, the pharmaceutical compositioncomprises embryonic-like placental stem cells of the invention and cordblood hematopoietic cells i.e., CD34+/CD38+ hematopoietic cells. One ormore of these cell populations can be used with or as a mixture withcord blood hematopoietic cells i.e., CD34+/CD38+ hematopoietic cells fortransplantation and other uses.

In one embodiment, the invention provides heterogeneous population ofnucleated cells that comprises embryonic-like placental stem cells. Incertain embodiments, a heterogeneous population of nucleated cells(rather than a pure population CD34+ cells embryonic-like placental stemcells) is preferred.

In another embodiment, the invention provides a mixed population ofcells (e.g., cord blood cells and embryonic-like placental stem cells).The population of mixed cells may be frozen or unfrozen. Such a mixedpopulation may be stored and/or used in one container, e.g., one bag orone syringe.

In another embodiment, the invention provides two or more separate ordistinct populations of different cell types (e.g., cord blood cells andembryonic-like placental stem cells). Each separate population may bestored and/or used in a separate container, e.g., one bag (e.g., bloodstorage bag from Baxter, Becton-Dickinson, Medcep, National HospitalProducts or Terumo) or one syringe, which contains a single type of cellor cell population.

In certain aspects of this embodiment, the invention provides separatecontainers of different cell types to be mixed before administration.Such cells may be unfrozen or frozen.

In a specific embodiment, cord blood cells are contained in one bag andembryonic-like placental stem cells are contained in a second bag. Inanother embodiment, the invention provides embryonic-like placental stemcells that are “conditioned” before freezing.

In another embodiment, a population of cells including, but not limitedto, embryonic-like placental stem cells may be conditioned by theremoval of red blood cells and/or granulocytes according to standardmethods, so that a population of nucleated cells remains that isenriched for embryonic-like placental stem cells. Such an enrichedpopulation of embryonic-like placental stem cells may be used unfrozen,or frozen for later use. If the population of cells is to be frozen, astandard cryopreservative (e.g., DMSO, glycerol, Epilife™ Cell FreezingMedium (Cascade Biologics)) is added to the enriched population of cellsbefore it is frozen.

In another embodiment, a population of cells including, but not limitedto, embryonic-like placental stem cells may be conditioned by theremoval of red blood cells and/or granulocytes after it has been frozenand thawed.

According to the invention, agents that induce cell differentiation maybe used to condition a population of embryonic-like stem cells. Incertain embodiments, an agent that induces differentiation can be addedto a population of cells within a container, including, but not limitedto, Ca²⁺, EGF, α-FGF, β-FGF, PDGF, keratinocyte growth factor (KGF),TGF-β, cytokines (e.g., IL-1α, IL-1β, IFN-γ, TFN), retinoic acid,transferrin, hormones (e.g., androgen, estrogen, insulin, prolactin,triiodothyronine, hydrocortisone, dexamethasone), sodium butyrate, TPA,DMSO, NMF, DMF, matrix elements (e.g., collagen, laminin, heparansulfate, Matrigel™), or combinations thereof.

In another embodiment, agents that suppress cellular differentiation canbe added to a population of embryonic-like stem cells. In certainembodiments, an agent that suppresses differentiation can be added to apopulation of cells within a container, including, but not limited to,human Delta-1 and human Serrate-1 polypeptides (see, Sakano et al., U.S.Pat. No. 6,337,387 entitled “Differentiation-suppressive polypeptide”,issued Jan. 8, 2002), leukemia inhibitory factor (LIF), stem cellfactor, or combinations thereof.

In certain embodiments, one or more populations of embryonic-like stemcells are delivered to a patient in need thereof. In certainembodiments, two or more populations of fresh (never frozen) cells aredelivered from a single container or single delivery system.

In another embodiment, two or more populations of frozen and thawedcells are delivered from a single container or single delivery system.

In another embodiment, each of two or more populations of fresh (neverfrozen) cells are transferred to, and delivered from, a single containeror single delivery system. In another embodiment, each of two or morepopulations of frozen and thawed cells are transferred to, and deliveredfrom, a single container or single delivery system. In another aspect ofthese embodiments, each population is delivered from a different IVinfusion bag (e.g., from Baxter, Becton-Dickinson, Medcep, NationalHospital Products or Terumo). The contents of each container (e.g., IVinfusion bag) may be delivered via a separate delivery system, or eachcontainer may be “piggybacked” so that their contents are combined ormixed before delivery from a single delivery system. For example, thetwo or more populations of cells may be fed into and/or mixed within acommon flow line (e.g., tubing), or they may be fed into and/or mixedwithin a common container (e.g., chamber or bag).

According to the invention, the two or more populations of cells may becombined before administration, during or at administration or deliveredsimultaneously.

In one embodiment, a minimum of 1.7×10⁷ nucleated cells/kg is deliveredto a patient in need thereof. Preferably, at least 2.5×10⁷ nucleatedcells/kg is delivered to a patient in need thereof.

In one embodiment, the invention provides a method of treating orpreventing a disease or disorder in a subject comprising administeringto a subject in which such treatment or prevention is desired atherapeutically effective amount of the embryonic-like stem cells, orsupplemented cell populations, of the invention.

In another embodiment, the invention provides a method of treating orpreventing a disease or disorder in a subject comprising administeringto a subject in which such treatment or prevention is desired atherapeutically effective amount of the embryonic-like stem cells of theinvention.

The embryonic-like stem cells of the invention are expected to have ananti-inflammatory effect when administered to an individual experiencinginflammation. In a preferred embodiment, the embryonic-like stem cellsor supplemental cell populations of the invention may be used to treatany disease, condition or disorder resulting from, or associated with,inflammation. The inflammation may be present in any organ or tissue,for example, muscle; nervous system, including the brain, spinal cordand peripheral nervous system; vascular tissues, including cardiactissue; pancreas; intestine or other organs of the digestive tract;lung; kidney; liver; reproductive organs; endothelial tissue, orendodermal tissue.

The embryonic-like stem cells or supplemented cell populations of theinvention may also be used to treat autoimmune or immune system-relateddisorders, including those associated with inflammation. Thus, incertain embodiments, the invention provides a method of treating anindividual having an autoimmune disease or condition, comprisingadministering to such individual a therapeutically effective amount ofthe cells or supplemented cell populations of the invention, whereinsaid disease or disorder can be, but is not limited to, diabetes,amylotrophic lateral sclerosis, myasthenia gravis, diabetic neuropathyor lupus. In related embodiments, the embryonic-like stem cells orsupplemented cell populations of the invention may be used to treatimmune-related disorders, such as chronic or acute allergies.

In certain embodiments, the disease or disorder includes, but is notlimited to, any of the diseases or disorders disclosed herein,including, but not limited to aplastic anemia, myelodysplasia,myocardial infarction, seizure disorder, multiple sclerosis, stroke,hypotension, cardiac arrest, ischemia, inflammation, age-related loss ofcognitive function, radiation damage, cerebral palsy, neurodegenerativedisease, Alzheimer's disease, Parkinson's disease, Leigh disease, AIDSdementia, memory loss, amyotrophic lateral sclerosis (ALS), ischemicrenal disease, brain or spinal cord trauma, heart-lung bypass, glaucoma,retinal ischemia, retinal trauma, lysosomal storage diseases, such asTay-Sachs, Niemann-Pick, Fabry's, Gaucher's, Hunter's, and Hurler'ssyndromes, as well as other gangliosidoses, mucopolysaccharidoses,glycogenoses, inborn errors of metabolism, adrenoleukodystrophy, cysticfibrosis, glycogen storage disease, hypothyroidism, sickle cell anemia,Pearson syndrome, Pompe's disease, phenylketonuria (PKU), porphyrias,maple syrup urine disease, homocystinuria, mucoplysaccharide nosis,chronic granulomatous disease and tyrosinemia, Tay-Sachs disease,cancer, tumors or other pathological or neoplastic conditions.

In other embodiments, the cells may be used in the treatment of any kindof injury due to trauma, particularly trauma involving inflammation.Examples of such trauma-related conditions include central nervoussystem (CNS) injuries, including injuries to the brain, spinal cord, ortissue surrounding the CNS injuries to the peripheral nervous system(PNS); or injuries to any other part of the body. Such trauma may becaused by accident, or may be a normal or abnormal outcome of a medicalprocedure such as surgery or angioplasty. The trauma may be related to arupture or occlusion of a blood vessel, for example, in stroke orphlebitis. In specific embodiments, the cells may be used in autologousor heterologous tissue regeneration or replacement therapies orprotocols, including, but not limited to treatment of corneal epithelialdefects, cartilage repair, facial dermabrasion, mucosal membranes,tympanic membranes, intestinal linings, neurological structures (e.g.,retina, auditory neurons in basilar membrane, olfactory neurons inolfactory epithelium), burn and wound repair for traumatic injuries ofthe skin, or for reconstruction of other damaged or diseased organs ortissues.

In a specific embodiment, the disease or disorder is aplastic anemia,myelodysplasia, leukemia, a bone marrow disorder or a hematopoieticdisease or disorder. In another specific embodiment, the subject is ahuman.

In another embodiment, the invention provides a method of treating anindividual having a disease, disorder or condition associated with orresulting from inflammation. In other embodiments, the inventionprovides a method of treating an individual having a neurologicaldisease, disorder or condition. In a more specific embodiment, saidneurological disease is ALS. In another more specific embodiment, saidneurological disease is Parkinson's disease. In another specificembodiment, said disease is a vascular or cardiovascular disease. In amore specific embodiment, said disease is atherosclerosis. In anotherspecific embodiment, said disease is diabetes.

In a specific embodiment, the pharmaceutical compositions of theinvention comprise an aliquot of umbilical cord blood to whichembryonic-like placental stem cells have been added as disclosed abovein Section 4.4.

A number of the embryonic-like stem cells, or of the supplemented cellpopulations, once administered, are able to engraft into the host,forming long-term “colonies.” This results in a host that is essentiallychimeric. Because chimeras in other genetic contexts are generally morevigorous and resilient, such chimerism is expected enhance the host'shealth and well-being. As such, the embryonic-like stem cells may beadministered not simply to an individual suffering from a specificdisease, disorder or condition, but may be administered to an individualin order to increase the individual's overall health and well-being.

The embryonic-like stem cells or supplemented cell populations of theinvention may be treated prior to administration to an individual withcompounds that modulate the activity of TNF-α. Such compounds aredisclosed in detail in copending U.S. Provisional Application No.60/372,348, filed Apr. 12, 2002, which disclosure is incorporated hereinin its entirety. Preferred compounds are referred to as IMiDs andSelCids, and particularly preferred compounds are available under thetrade names Actimid™ and Revimid™.

A particularly useful aspect of the embryonic-like stem cells of theinvention is that, in certain embodiments, there is no need to HLA-typethe cells prior to administration. In other words, embryonic-like stemcells may be taken from a heterologous donor, or a plurality ofheterologous donors, and transplanted to an individual in need of suchcells, and the transplanted cells will remain within the hostindefinitely. This elimination of the need for HLA typing greatlyfacilitates both the transplantation procedure itself and theidentification of donors for transplantation. However, theembryonic-like stem cells or supplemented cell populations containingthem may be HLA matched (donor to recipient) prior to administration.

The inventors have discovered that the efficacy of treating anindividual with the embryonic-like stem cells or supplemented cellpopulations is enhanced if these cells are preconditioned.Preconditioning comprises storing the cells in a gas-permeable containerof a period of time at approximately −5 to 23° C., 0-10° C., orpreferably 4-5° C. The period of time may be between 18 hours and 21days, between 48 hours and 10 days, and is preferably between 3-5 days.The cells may be cryopreserved prior to preconditioning or, preferably,are preconditioned immediately prior to administration.

Thus, in one embodiment, the invention provides a method of treating anindividual comprising administering to said individual embryonic-likestem cells collected from at least one donor. “Donor” as used hereinmeans an adult, child, infant, or, preferably, a placenta. In another,preferred, embodiment, the method comprises administering to saidindividual embryonic-like stem cells that are collected from a pluralityof donors and pooled. In a specific embodiment, said embryonic-like stemcells are stem cells taken from a plurality of donors. When collectedform multiple donors, the dosage units, where a “dosage unit” is acollection from a single donor, may be pooled prior to administration,may be administered sequentially, or may be administered alternatively.In another embodiment of the method, said embryonic-like stem cells aremixed with, or “spiked” into umbilical cord blood, and the mixtureadministered to an individual. In more specific embodiments of themethod, the ratio of embryonic-like stem cells to cord blood may be atleast 20:80, 30:70, 40:60, 50:50, 60:40, 70:30 or 80:20, by number oftotal nucleated cells.

4.7 Administration of Stem Cells: Dosages

A particularly useful aspect of the invention is the administration ofhigh doses of stem cells to an individual; such numbers of cells aresignificantly more effective than the material (for example, bone marrowor cord blood) from which they were derived. In this context, “highdose” indicates 5, 10, 15 or 20 or more times the number of totalnucleated cells, including stem cells, particularly embryonic-like stemcells, than would be administered, for example, in a bone marrowtransplant. Typically, a patient receiving a stem cell infusion, forexample for a bone marrow transplantation, receives one unit of cells,where a unit is approximately 1×10⁹ nucleated cells (corresponding to1-2×10⁸ stem cells). For high-dose therapies, therefore, a patient wouldbe administered 3 billion, 5 billion, 10 billion, 15 billion, 20billion, 30 billion, 40 billion, 50 billion or more, or, alternatively,3, 5, 10, 20, 30, 40, or 50 units or more, of total nucleated cells,either embryonic-like stem cells alone, or embryonic-like stem cellsspiked into another stem or progenitor cell population (e.g.,embryonic-like stem cells spiked into umbilical cord blood). In onepreferred embodiment, for example, an individual is given 15 units ofspiked cord blood, where the unit contains approximately 750 millioncord blood cells and 500 million embryonic-like stem cells. Thus, in oneembodiment, the number of nucleated cells administered to an individualis at least five times the number of cells normally administered in abone marrow replacement. In another specific embodiment of the method,the number of nucleated cells administered to an individual is at leastten times the number of cells normally administered in a bone marrowreplacement. In another specific embodiment of the method, the number ofnucleated cells administered to an individual is at least fifteen timesthe number of cells normally administered in a bone marrow replacement.In another embodiment of the method, the total number of nucleatedcells, which includes stem cells, administered to an individual isbetween 1-100×10⁸ per kilogram of body weight. In another embodiment,the number of total nucleated cells administered is at least 5 billioncells. In another embodiment, the total number of nucleated cellsadministered is at least 15 billion cells.

In another embodiment of the method, said embryonic-like stem cells andsaid cord blood are mixed immediately prior to (i.e., within fiveminutes of) administration to said individual. In another embodiment,said embryonic-like stem cells and said cord blood are mixed at a pointin time more than five minutes prior to administration to saidindividual. In another embodiment of the method, the embryonic-like stemcells are cryopreserved and thawed prior to administration to saidindividual. In another embodiment, said embryonic-like stem cells andsaid cord blood are mixed to form a supplemented cell population at apoint in time more than twenty-four hours prior to administration tosaid individual, wherein said supplemented cell population has beencryopreserved and thawed prior to said administration. In anotherembodiment, said embryonic-like stem cells and/or supplemented cellpopulations may be administered more than once. In another embodiment,said embryonic-like stem cells and/or supplemented cell populations arepreconditioned by storage from between 18 hours and 21 days prior toadministration. In a more specific embodiment, the cells arepreconditioned for 48 hours to 10 days prior to administration. In apreferred specific embodiment, said cells are preconditioned for 3-5days prior to transplantation. In a preferred embodiment of any of themethods herein, said embryonic-like stem cells are not HLA typed priorto administration to an individual.

In another specific embodiment of the method, said embryonic-like stemcells are primarily (i.e., >50%) CD34+ cells. In a more specificembodiment of the method, said embryonic-like stem cells are primarilyCD34+33+ stem cells.

Therapeutic or prophylactic treatment of an individual withembryonic-like stem cells or supplemented cell populations containingthem may be considered efficacious if the disease, disorder or conditionis measurably improved in any way. Such improvement may be shown by anumber of indicators. Measurable indicators include, for example,detectable changes in a physiological condition or set of physiologicalconditions associated with a particular disease, disorder or condition(including, but not limited to, blood pressure, heart rate, respiratoryrate, counts of various blood cell types, levels in the blood of certainproteins, carbohydrates, lipids or cytokines or modulation expression ofgenetic markers associated with the disease, disorder or condition).Treatment of an individual with the stem cells or supplemented cellpopulations of the invention would be considered effective if any one ofsuch indicators responds to such treatment by changing to a value thatis within, or closer to, the normal value. The normal value may beestablished by normal ranges that are known in the art for variousindicators, or by comparison to such values in a control. In medicalscience, the efficacy of a treatment is also often characterized interms of an individual's impressions and subjective feeling of theindividual's state of health. Improvement therefore may also becharacterized by subjective indicators, such as the individual'ssubjective feeling of improvement, increased well-being, increased stateof health, improved level of energy, or the like, after administrationof the stem cells or supplemented cell populations of the invention.

The embryonic-like stem cells and supplemented cell populations of theinvention may be administered to a patient in any pharmaceutically ormedically acceptable manner, including by injection or transfusion. Thecells or supplemented cell populations may be contain, or be containedin any pharmaceutically-acceptable carrier (See Section 4.8). Theembryonic-like stem cells or supplemented cell populations may becarried, stored, or transported in any pharmaceutically or medicallyacceptable container, for example, a blood bag, transfer bag, plastictube or vial.

4.7. Kits

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be: an apparatus for cell culture, one or morecontainers filled with a cell culture medium or one or more componentsof a cell culture medium, an apparatus for use in delivery of thecompositions of the invention, e.g., an apparatus for the intravenousinjection of the compositions of the invention, and/or a notice in theform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. In a specific embodiment, the kit comprises one or morecontainers filled with embryonic-like stem cells of the invention andone or more different containers filled with stem cells, e.g., umbilicalcord blood, as disclosed above.

In one embodiment, the kit comprises a mixture of stem cells, e.g., cordblood cells, supplemented with embryonic-like stem cells containedwithin one bag or container. In another embodiment, the kit comprises apopulation of cord blood cells and a population of embryonic-like stemcells that are contained within two separate bags or containers. Incertain embodiments, the kit comprises a “two bag” composition whereinthe bag containing the cord blood cells and the bag containing theembryonic-like stem cells is mixed prior to, or at the time of,administration to a patient in need thereof. In other embodiments, thekit comprises a population of cord blood cells and a population ofembryonic-like stem cells that are contained within two separate bags orcontainers and that are administered separately (e.g., simultaneously orsequentially) to a patient, wherein the mixing of the two cellpopulations occurs in vivo.

In another embodiment, the kit provides a population of cord blood cellsand a population of embryonic-like stem cells that are physically mixedprior to administration. In another aspect of this embodiment, the kitcomprises a container containing a growth factor, e.g., GM-CSF, IL-4,Flt3L, CD40L, IFN-alpha, TNF-alpha, IFN-gamma, IL-2, IL-6, retinoicacid, basic fibroblast growth factor, TGF-beta-1, TGF-beta-3, hepatocytegrowth factor, epidermal growth factor, cardiotropin-1, angiotensinogen,angiotensin I (AI), angiotensin II (AII), AII AT₂ type 2 receptoragonists, or analogs or fragments thereof. In another aspect of thisembodiment, the two populations are physically mixed and then treatedwith the growth factor comprised in the kit, to induce celldifferentiation, prior to administration to the patient. In anotheraspect of this embodiment, the cord blood cells and/or theembryonic-like stem cells are treated with the growth factor comprisedin the kit, to induce cell differentiation and then physically mixedprior to administration to the patient.

The following experimental examples are offered by way of illustrationand not by way of limitation.

5. EXAMPLES 5.1. Example 1 Analysis of Cell Types Recovered fromPerfusate of Drained Placenta

This example describes the analysis of the cell types recovered from theeffluent perfusate of a placenta cultured according to the methods ofthe invention.

Twenty ml of phosphate buffered saline solution (PBS) was added to theperfusion liquid and a 10 ml portion was collected and centrifuged for25 minutes at 3000 rpm (revolutions per minute). The effluent wasdivided into four tubes and placed in an ice bath. 2.5 ml of a 1% fetalcalf serum (FCS) solution in PBS was added and the tubes werecentrifuged (140 minutes×10 g (acceleration due to gravity)). The pelletwas resuspended in 5 ml of 1% FCS and two tubes were combined. The totalmononucleocytes were calculated by adding the total lymphocytes and thetotal monocytes, and then multiplying the result by the total cellsuspension volume.

The following table discloses the types of cells obtained by perfusionof a cultured placenta according to the methods described hereinabove.

WBC Total # of 1000/ml Lym % MID % GRA % Volume Cells CB 10.5 43.2  848.8 60 ml 6.3 × 10⁸ (Cord Blood) PP 12.0 62.9 18.2 18.9 15 ml 1.8 × 10⁸(Placenta perfusate, room temperature) PP₂ 11.7 56.0 19.2 24.8 30 ml 3.5× 10⁸ (Placenta perfusate, 37° C.) Samples of PP were after Ficoll.Total cell number of PP after Ficoll was 5.3 × 10⁸ and number of CBbefore processing is 6.3 × 10⁸. Lym % indicates percent of lymphocytes;MID % indicates percent of midrange white blood cells; and GRA %indicates percent of granulocytes.

5.2. Example 2 Analysis of Cells Obtained by Perfusion and Incubation ofPlacenta

The following example describes an analysis of cells obtained byperfusion and incubation of placenta according to the methods of theinvention.

5.2.1. Materials and Methods

Placenta donors were recruited from expectant mothers that enrolled inprivate umbilical cord blood banking programs and provided informedconsent permitting the use of the exsanguinated placenta followingrecovery of cord blood for research purposes. Donor data may beconfidential. These donors also permitted use of blinded data generatedfrom the normal processing of their umbilical cord blood specimens forcryopreservation. This allowed comparison between the composition of thecollected cord blood and the effluent perfusate recovered using theexperimental method described below.

Following exsanguination of cord blood from the umbilical cord andplacenta is stored at room temperature and delivered to the laboratorywithin four to twenty-four hour, according to the methods describedhereinabove, the placenta was placed in a sterile, insulated containerat room temperature and delivered to the laboratory within 4 hours ofbirth. Placentas were discarded if, on inspection, they had evidence ofphysical damage such as fragmentation of the organ or avulsion ofumbilical vessels. Placentas were maintained at room temperature (23±2°C.) or refrigerated (4° C.) in sterile containers for 2 to 20 hours.Periodically, the placentas were immersed and washed in sterile salineat 25±3° C. to remove any visible surface blood or debris.

The umbilical cord was transected approximately 5 cm from its insertioninto the placenta and the umbilical vessels were cannulated with TEFLON®or polypropylene catheters connected to a sterile fluid path allowingbi-directional perfusion of the placenta and recovery of the effluentfluid. The methods described hereinabove enabled all aspects ofplacental conditioning, perfusion and effluent collection to beperformed under controlled ambient atmospheric conditions as well asreal-time monitoring of intravascular pressure and flow rates, core andperfusate temperatures and recovered effluent volumes. A range ofconditioning protocols were evaluated over a 24-hour postpartum period,and the cellular composition of the effluent fluid was analyzed by flowcytometry, light microscopy and colony forming unit assays.

5.2.2. Placental Conditioning

The donor placentas were processed at room temperature within 12 to 24hours after delivery. Before processing, the membranes were removed andthe maternal site washed clean of residual blood. The umbilical vesselswere cannulated with catheters made from 20 gauge Butterfly needles usefor blood sample collection.

The donor placentas were maintained under varying conditions such asmaintenance at 5-37° 5% CO₂, pH 7.2 to 7.5, preferably pH 7.45, in anattempt to simulate and sustain a physiologically compatible environmentfor the proliferation and recruitment of residual embryonic-like stemcells. The cannula was flushed with IMDM serum-free medium (GibcoBRL,NY) containing 2U/ml heparin (Elkins-Sinn, N.J.). Perfusion of theplacenta continued at a rate of 50 ml per minute until approximately 150ml of perfusate was collected. This volume of perfusate was labeled“early fraction.” Continued perfusion of the placenta at the same rateresulted in the collection of a second fraction of approximately 150 mland was labeled “late fraction.” During the course of the procedure, theplacenta was gently massaged to aid in the perfusion process and assistin the recovery of cellular material. Effluent fluid was collected fromthe perfusion circuit by both gravity drainage and aspiration throughthe arterial cannula.

Placentas were then perfused with heparinized (2U/ml) Dulbecco'smodified Eagle Medium (H.DMEM) at the rate of 15 ml/minute for 10minutes and the perfusates were collected from the maternal sites withinone hour and the nucleated cells counted. The perfusion and collectionprocedures were repeated once or twice until the number of recoverednucleated cells fell below 100/ml. The perfusates were pooled andsubjected to light centrifugation to remove platelets, debris andde-nucleated cell membranes. The nucleated cells were then isolated byFicoll-Hypaque density gradient centrifugation and after washing,resuspended in H.DMEM. For isolation of the adherent cells, aliquots of5-10×10⁶ cells were placed in each of several T-75 flasks and culturedwith commercially available Mesenchymal Stem Cell Growth Medium (MSCGM)obtained from BioWhittaker, and placed in a tissue culture incubator(37° C., 5% CO₂). After 10 to 15 days, the non-adherent cells wereremoved by washing with PBS, which was then replaced by MSCGM. Theflasks were examined daily for the presence of various adherent celltypes and in particular, for identification and expansion of clusters offibroblastoid cells.

5.2.3. Cell Recovery and Isolation

Cells were recovered from the perfusates by centrifugation at 5000×g for15 minutes at room temperature. This procedure served to separate cellsfrom contaminating debris and platelets. The cell pellets wereresuspended in IMDM serum-free medium containing 2U/ml heparin and 2 mMEDTA (GibcoBRL, NY). The total mononuclear cell fraction was isolatedusing Lymphoprep (Nycomed Pharma, Oslo, Norway) according to themanufacturer's recommended procedure and the mononuclear cell fractionwas resuspended. Cells were counted using a hemocytometer. Viability wasevaluated by trypan blue exclusion. Isolation of mesenchymal cells wasachieved by “differential trypsinization,” using a solution of 0.05%trypsin with 0.2% EDTA (Sigma, St. Louis Mo.). Differentialtrypsinization was possible because fibroblastoid cells detached fromplastic surfaces within about five minutes whereas the other adherentpopulations required more than 20-30 minutes incubation. The detachedfibroblastoid cells were harvested following trypsinization and trypsinneutralization, using Trypsin Neutralizing Solution (TNS, BioWhittaker).The cells were washed in H.DMEM and resuspended in MSCGM.

Flow cytometry was carried out using a Becton-Dickinson FACSCaliburinstrument and FITC and PE labeled monoclonal antibodies (mAbs),selected on the basis of known markers for bone marrow-derived MSC(mesenchymal stem cells), were purchased from B. D. and Caltaglaboratories (South San Francisco, Calif.), and SH2, SH3 and SH4antibody producing hybridomas were obtained from and reactivities of themAbs in their cultured supernatants were detected by FITC or PE labeledF(ab)′² goat anti-mouse antibodies. Lineage differentiation was carriedout using commercially available induction and maintenance culture media(BioWhittaker), used as per manufacturer's instructions.

5.2.4. Isolation of Placental Embryonic-Like Stem Cells

Microscopic examination of the adherent cells in the culture flasksrevealed morphologically different cell types. Spindle-shaped cells,round cells with large nuclei and numerous perinuclear small vacuoles,and star-shaped cells with several projections (through one of whichstar-shaped cells were attached to the flask) were observed adhering tothe culture flasks. Although no attempts were made to furthercharacterize these adherent cells, similar cells were observed in theculture of bone marrow, cord and peripheral blood, and thereforeconsidered to be non-stem cell-like in nature. The fibroblastoid cells,appearing last as clusters, were candidates for being MSC (mesenchymalstem cells) and were isolated by differential trypsinization andsubcultured in secondary flasks. Phase microscopy of the rounded cells,after trypsinization, revealed that the cells were highly granulated;indistinguishable from the bone marrow-derived MSC produced in thelaboratory or purchased from BioWhittaker. When subcultured, theplacenta-derived embryonic-like stem cells, in contrast to their earlierphase, adhered within hours, assumed characteristic fibroblastoid shape,and formed a growth pattern identical to the reference bonemarrow-derived MSC. During subculturing and refeeding, moreover, theloosely bound mononuclear cells were washed out and the culturesremained homogeneous and devoid of any visible non-fibroblastoid cellcontaminants.

5.2.5. Results

The expression of CD-34, CD-38, and other stem cell-associated surfacemarkers on early and late fraction purified mononuclear cells wasassessed by flow cytometry. Recovered, sorted cells were washed in PBSand then double-stained with antiCD34 phycoerythrin and anti-CD38fluorescein isothiocyanate (Becton Dickinson, Mountain View, Calif.).

Cell isolation was achieved by using magnetic cell separation, such asfor example, Auto Macs (Miltenyi). Preferably, CD 34+ cell isolation isperformed first.

5.3. Example 3 Perfusion Medium

The following example provides a formula of the preferred perfusatesolution for the cultivation of isolated placentas.

Stock Final Chemical Source Concentration Concentration 500 ml DMEM-LGGibcoBRL11885- 300 ml 084 MCDB201 Sigma M-6770 dissolved in pH to 7.2.200 ml H2O filter FCS Hyclone 100% 2%  10 ml ITS Sigma I-3146 or 100x 1x 5 ml GibcoBRL41400- 045 Pen & Strep GibcoBRL15140- 100x 1x  5 ml 122LA + BSA Sigma + GibcoBRL 100x(1 μg/ml of 10 ng/ml of LA  5 ml BSA LADexamethasone Sigma D-2915 0.25 mM in H2O 0.05 μM 100 μl L-AscorbicSigma A-8960 1000x(100 mM) 1x(0.1 mM) 500 μl Acid PDGF (50 μg) R&D 220BD10 μg/ml in 10 ng/ml 500 μl 4 mM HCl + 0.1% BSA EGF (200 μg) SigmaE-9644 10 μg/ml in 10 ng/ml 500 μl 10 mM HAc + 0.1% BSA

The above-composition is a perfusate that may be used at a variety oftemperatures to perfuse placenta. It should be noted that additionalcomponents such as antibiotics, anticoagulant and other growth factorsmay be used in the perfusate or culture media.

5.4 Example 4 Induction of Differentiation into Particular Cell Types

Cord blood cells and/or embryonic-like stem cells are induced todifferentiate into a particular cell type by exposure to a growthfactor. Growth factors that are used to induce induction include, butare not limited to: GM-CSF, IL-4, Flt3L, CD40L, IFN-alpha, TNF-alpha,IFN-gamma, IL-2, IL-6, retinoic acid, basic fibroblast growth factor,TGF-beta-1, TGF-beta-3, hepatocyte growth factor, epidermal growthfactor, cardiotropin-1, angiotensinogen, angiotensin I (AI), angiotensinII (AII), AII AT₂ type 2 receptor agonists, or analogs or fragmentsthereof.

5.4.1 Induction Of Differentiation Into Neurons

This example describes the induction of cord blood cells and/orembryonic-like stem cells to differentiate into neurons. The followingprotocol is employed to induce neuronal differentiation:

-   1. Placental stem cells are grown for 24 hr in preinduction media    consisting of DMEM/20% FBS and 1 mM beta-mercaptoethanol.-   2. Preinduction media is removed and cells are washed with PBS.-   3. Neuronal induction media consisting of DMEM and 1-10 mM    betamercaptoethanol is added. Alternatively, induction media    consisting of DMEM/2% DMSO/200 μM butylated hydroxyanisole may be    used to enhance neuronal differentiation efficiency.-   4. In certain embodiments, morphologic and molecular changes may    occur as early as 60 minutes after exposure to serum-free media and    betamercaptoethanol (Woodbury et al., J. Neurosci. Res.,    61:364-370). RT/PCR may be used to assess the expression of e.g.,    nerve growth factor receptor and neurofilament heavy chain genes.

5.4.2 Induction Of Differentiation Into Adipocytes

This example describes the induction of cord blood cells and/orembryonic-like stem cells to differentiate into adipocytes. Thefollowing protocol is employed to induce adipogenic differentiation:

-   1. Placental stem cells are grown in MSCGM (Bio Whittaker) or DMEM    supplemented with 15% cord blood serum.-   2. Three cycles of induction/maintenance are used. Each cycle    consists of feeding the placental stem cells with Adipogenesis    Induction Medium (Bio Whittaker) and culturing the cells for 3 days    (at 37° C., 5% CO₂), followed by 1-3 days of culture in Adipogenesis    Maintenance Medium (Bio Whittaker). An induction medium is used that    contains 1 μM dexamethasone, 0.2 mM indomethacin, 0.01 mg/ml    insulin, 0.5 mM IBMX, DMEM-high glucose, FBS, and antibiotics.-   3. After 3 complete cycles of induction/maintenance, the cells are    cultured for an additional 7 days in adipogenesis maintenance    medium, replacing the medium every 2-3 days.-   4. Adipogenesis may be assessed by the development of multiple    intracytoplasmic lipid vesicles that can be easily observed using    the lipophilic stain oil red O. RT/PCR assays are employed to    examine the expression of lipase and fatty acid binding protein    genes.

5.4.3 Induction of Differentiation into Chondrocytes

This example describes the induction of cord blood cells and/orembryonic-like stem cells to differentiate into chondrocytes. Thefollowing protocol is employed to induce chondrogenic differentiation:

-   1. Placental stem cells are maintained in MSCGM (Bio Whittaker) or    DMEM supplemented with 15% cord blood serum.-   2. Placental stem cells are aliquoted into a sterile polypropylene    tube. The cells are centrifuged (150×g for 5 minutes), and washed    twice in Incomplete Chondrogenesis Medium (Bio Whittaker).-   3. After the last wash, the cells are resuspended in Complete    Chondrogenesis Medium (Bio Whittaker) containing 0.01 μg/ml    TGF-beta-3 at a concentration of 5×10(5) cells/ml.-   4. 0.5 ml of cells is aliquoted into a 15 ml polypropylene culture    tube. The cells are pelleted at 150×g for 5 minutes. The pellet is    left intact in the medium.-   5. Loosely capped tubes are incubated at 37° C., 5% CO₂ for 24    hours.-   6. The cell pellets are fed every 2-3 days with freshly prepared    complete chondrogenesis medium.-   7. Pellets are maintained suspended in medium by daily agitation    using a low speed vortex.-   8. Chondrogenic cell pellets are harvested after 14-28 days in    culture.-   9. Chondrogenesis may be characterized by e.g., observation of    production of esoinophilic ground substance, assessing cell    morphology, an/or RT/PCR for examining collagen 2 and collagen 9    gene expression.

5.4.4 Induction of Differentiation into Osteocytes

This example describes the induction of cord blood cells and/orembryonic-like stem cells to differentiate into osteocytes. Thefollowing protocol is employed to induce osteogenic differentiation:

-   1. Adherent cultures of placental stem cells are cultured in MSCGM    (Bio Whittaker) or DMEM supplemented with 15% cord blood serum.-   2. Cultures are rested for 24 hours in tissue culture flasks.-   3. Osteogenic differentiation is induced by replacing MSCGM with    Osteogenic Induction Medium (Bio Whittaker) containing 0.1 μM    dexamethasone, 0.05 mM ascorbic acid-2-phosphate, 10 mM beta    glycerophosphate.-   4. Cells are fed every 3-4 days for 2-3 weeks with Osteogenic    Induction Medium.-   5. Differentiation is assayed using a calcium-specific stain and    RT/PCR for alkaline phosphatase and osteopontin gene expression.

5.4.5 Induction Of Differentiation Into Hepatocytes

This example describes the induction of cord blood cells and/orembryonic-like stem cells to differentiate into hepatocytes. Thefollowing protocol is employed to induce hepatogenic differentiation:

-   1. Placental stem cells are cultured in DMEM/20% CBS supplemented    with hepatocyte growth factor, 20 ng/ml; and epidermal growth    factor, 100 ng/ml. KnockOut Serum Replacement may be used in lieu of    FBS.-   2. IL-6 50 ng/ml is added to induction flasks.

5.4.6 Induction of Differentiation into Pancreatic Cells

This example describes the induction of cord blood cells and/orembryonic-like stem cells to differentiate into pancreatic cells. Thefollowing protocol is employed to induce pancreatic differentiation:

-   1. Placental stem cells are cultured in DMEM/20% CBS, supplemented    with basic fibroblast growth factor, 10 ng/ml; and transforming    growth factor beta-1, 2 ng/ml. KnockOut Serum Replacement may be    used in lieu of CBS.-   2. Conditioned media from nestin-positive neuronal cell cultures is    added to media at a 50/50 concentration.-   3. Cells are cultured for 14-28 days, refeeding every 3-4 days.-   4. Differentiation is characterized by assaying for insulin protein    or insulin gene expression by RT/PCR.

5.4.7 Induction of Differentiation into Cardiac Cells

This example describes the induction of cord blood cells and/orembryonic-like stem cells to differentiate into cardiac cells. Thefollowing protocol is employed to induce myogenic differentiation:

-   1. Placental stem cells are cultured in DMEM/20% CBS, supplemented    with retinoic acid, 1 μM; basic fibroblast growth factor, 10 ng/ml;    and transforming growth factor beta-1, 2 ng/ml; and epidermal growth    factor, 100 ng/ml. KnockOut Serum Replacement may be used in lieu of    CBS.-   2. Alternatively, placental stem cells are cultured in DMEM/20% CBS    supplemented with 50 ng/ml Cardiotropin-1 for 24 hours.-   3. Alternatively, placental stem cells are maintained in    protein-free media for 5-7 days, then stimulated with human    myocardium extract (escalating dose analysis). Myocardium extract is    produced by homogenizing 1 gm human myocardium in 1% HEPES buffer    supplemented with 1% cord blood serum. The suspension is incubated    for 60 minutes, then centrifuged and the supernatant collected.-   4. Cells are cultured for 10-14 days, refeeding every 3-4 days.-   5. Differentiation is assessed using cardiac actin RT/PCR gene    expression assays.

5.4.8 Characterization of Cord Blood Cells and/or Embryonic-Like StemCells Prior to and/or After Differentiation

The embryonic-like stem cells, the cord blood cells and/or thepopulations of cord blood cells spiked with embryonic-like stem cellsare characterized prior to and/or after differentiation by measuringchanges in morphology and cell surface markers using techniques such asflow cytometry and immunocytochemistry, and measuring changes in geneexpression using techniques, such as PCR. Cells that have been exposedto growth factors and/or that have differentiated are characterized bythe presence or absence of the following cell surface markers: CD10+,CD29+, CD34−, CD38−, CD44+, CD45−, CD54+, CD90+, SH2+, SH3+, SH4+,SSEA3−, SSEA4−, OCT-4+, and ABC-p+. Preferably, the embryonic-like stemcell are characterized, prior to differentiation, by the presence ofcell surface markers OCT-4+, APC-p+, CD34− and CD38−. Stem cells bearingthese markers are as versatile (e.g., pluripotent) as human embryonicstem cells. Cord blood cells are characterized, prior todifferentiation, by the presence of cell surface markers CD34+ andCD38+. Differentiated cells derived from embryonic-like stem cells, cordblood cells and/or a populations of cord blood cells spiked withembryonic-like stem cells preferably do not express these markers.

5.5 Example 5 Treatment of Individuals Having Amylotrophic LateralSclerosis with Embryonic-Like Stem Cells

Amyotrophic Lateral Sclerosis (ALS), also called Lou Gehrig's disease,is a fatal neurodegenerative disease affecting motor neurons of thecortex, brain stem and spinal cord. ALS affects as many as 20,000Americans with 5,000 new cases occurring in the US each year. Themajority of ALS cases are sporadic (S-ALS) while 5-10% are hereditary(familial—F-ALS). ALS occurs when specific nerve cells in the brain andspinal cord that control voluntary movement gradually degenerate. Thecardinal feature of ALS is the loss of spinal motor neurons which causesthe muscles under their control to weaken and waste away leading toparalysis. ALS manifests itself in different ways, depending on whichmuscles weaken first. ALS strikes in mid-life with men beingone-and-a-half times more likely to have the disease as women. ALS isusually fatal within five years after diagnosis.

ALS has both familial and sporadic forms, and the familial forms havenow been linked to several distinct genetic loci. Only about 5-10% ofALS cases are familial. Of these, 15-20% are due to mutations in thegene encoding Cu/Zn superoxide dismutase 1 (SOD1). These appear to be“gain-of-function” mutations that confer toxic properties on the enzyme.The discovery of SOD mutations as a cause for ALS has paved the way forsome progress in the understanding of the disease; animal models for thedisease are now available and hypotheses are being developed and testedconcerning the molecular events leading to cell death.

Presented below is an example method of treating an individual havingALS with embryonic-like stem cells derived from placenta. The methodinvolves intravenous infusion through a peripheral, temporaryangiocatheter.

An individual having ALS is first assessed by the performance ofstandard laboratory analyses. Such analyses may include a metabolicprofile; CDC with differential; lipid profile; fibrinogen level; ABO rHtyping of the blood; liver function tests; and determination ofBUN/creatine levels. Individuals are instructed the day prior to thetransplant to take the following medications: diphenhydramine(Benadryl™), 25 mg t.i.d, and prednisone, 10 mg.

The embryonic-like stem cells, either alone or spiked into cord blood,are taken from cryopreserved stock, thawed, and maintained forapproximately two days prior to transplantation at a temperature ofapproximately 5° C.

The individual is transplanted at an outpatient clinical center whichhas all facilities necessary for intravenous infusion, physiologicalmonitoring and physical observation. Approximately one hour prior totransplantation, the individual receives diphenhydramine (Benadryl™), 25mg×1 P.O., and prednisone, 10 mg×1 P.O. This is precautionary, and ismeant to reduce the likelihood of an acute allergic reaction. At thetime of transfusion, an 18 G indwelling peripheral venous line is placesinto one of the individual's extremities, and is maintained open byinfusion of D5 ½ normal saline +20 mEq KCl at a TKO rate. The individualis examined prior to transplantation, specifically to note heart rate,respiratory rate, temperature. Other monitoring may be performed, suchas an electrocardiogram and blood pressure measurement.

Embryonic-like stem cells are then infused at a rate of 1 unit per hourin a total delivered fluid volume of 60 ml, where a unit isapproximately 1-2 10⁹ total nucleated cells. Alternatively, the unit ofembryonic-like stem cells is delivered in cord blood having a totalfluid volume of 60 ml. In this case, the ratio of the number ofembryonic-like stem cells to stem cells in the cord blood is at least2:1. The administered unit may also consist of cord blood alone. Basedupon data from pre-clinical studies in mice, a total of 2.0-2.5×10⁸cells per kilogram of body weight should be administered. For example, a70 kilogram individual would receive approximately 14-18×10⁹ totalnucleated cells. The individual should be monitored for signs ofallergic response or hypersensitivity, which are signals for immediatecessation of infusion.

Post-infusion, the individual should be monitored in a recumbentposition for at least 60 minutes, whereupon he or she may resume normalactivities.

5.6 Example 6 Treatment of Individuals Having Atherosclerosis UsingEmbryonic-Like Stem Cells

The infusion protocol outlined in Example 5 may be used to administerthe embryonic-like stem cells, either alone or spiked into umbilicalcord blood, to a patient having atherosclerosis. The embryonic-like stemcells or supplemented cell populations may be administered toasymptomatic individuals, individuals that are candidates forangioplasty, or to patients that have recently (within one week)undergone cardiac surgery.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention.

1. A composition comprising human stem or progenitor cells and isolatedhuman placental stem cells that are SH2⁺, SH3⁺, SH4⁺ and OCT-4⁺, whereinsaid placental stem cells are obtained from a placenta that has beendrained of cord blood and flushed to remove residual blood.
 2. Acomposition comprising human umbilical cord blood cells and isolatedhuman placental stem cells that are SH2⁺, SH3⁺, SH4⁺ and OCT-4⁺, whereinsaid placental stem cells are obtained from a placenta that has beendrained of cord blood and flushed to remove residual blood.
 3. Acomposition comprising human stem or progenitor cells and isolated humanplacental stem cells that are SH2⁺, SH3⁺, SH4⁺, SSEA3⁻ and SSEA4⁻,wherein said placental stem cells are obtained from a placenta that hasbeen drained of cord blood and flushed to remove residual blood.
 4. Acomposition comprising human umbilical cord blood cells and isolatedhuman placental stem cells that are SH2⁺, SH3⁺, SH4⁺, SSEA3⁻ and SSEA4⁻,wherein said placental stem cells are obtained from a placenta that hasbeen drained of cord blood and flushed to remove residual blood.
 5. Thecomposition of any of claim 1, 2, 3 or 4 that is contained in acontainer.
 6. The composition of claim 5 wherein the container issealed, air tight, and sterile.
 7. The composition of claim 1 or claim 3wherein the stem or progenitor cells are from umbilical cord blood orplacental blood, fetal or neonatal hematopoietic stem or progenitorcells, adult cells or bone marrow stem or progenitor cells.
 8. Thecomposition of claim 7 wherein the stem or progenitor cells are fetal orneonatal hematopoietic stem or progenitor cells.
 9. The composition ofclaim 8 wherein a plurality of the hematopoietic stem or progenitorcells are CD34⁺ and CD38⁻.
 10. The composition of claim 2 or claim 4wherein a plurality of the umbilical cord blood stem cells are CD34⁺ andCD38⁺.
 11. The composition of claim 2 or claim 4 wherein a plurality ofthe umbilical cord blood stem cells are CD34⁺ and CD38⁺.
 12. Thecomposition of claim 1 or 2 wherein the placental stem cells exhibit atleast one of the following cell surface markers: CD10, CD29, CD44, CD54,or CD90, or lack at least one of the following cell surface markers:CD34, CD45, SSEA3, or SSEA4.
 13. A kit comprising a composition of claim1 or 3 wherein said stem or progenitor cells are in a first containerand said placental stem cells are in a second container.
 14. Acomposition which comprises human stem or progenitor cells fromumbilical cord blood supplemented with a plurality of isolated humanplacental stem cells that are SH2⁺, SH3⁺, SH4⁺ and OCT-4⁺, wherein saidplacental stem cells are obtained from a placenta that has been drainedof cord blood and flushed to remove residual blood, and wherein theratio of placental stem cells to said stem or progenitor cells is atleast 1:1 in numbers.
 15. The composition of claim 3 or 4 wherein theplacental stem cells exhibit at least one of the following cell surfacemarkers: CD10, CD29, CD44, CD54, CD90, or OCT-4, or lack at least one ofthe following cell surface markers: CD34 or CD45.
 16. A kit comprising acomposition of claim 2 or claim 4 wherein said umbilical cord bloodcells are in a first container and said placental stem cells are in asecond container.
 17. A composition which comprises a number of humanstem or progenitor cells from umbilical cord blood supplemented with anumber of isolated human placental stem cells that are SH2⁺, SH3⁺, SH4⁺,SSEA3⁻ and SSEA4³¹ , wherein said placental stem cells are obtained froma placenta that has been drained of cord blood and flushed to removeresidual blood, and wherein the ratio of the number of placental stemcells to the number of said stem or progenitor cells is at least 1:1.